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Abstract:

A method of diagnosing a disease in which an miRNA is expressed at a lower
level comprises, in a test sample obtained from a subject, determining
the methylation status of at least one gene encoding the miRNA in the
sample, wherein the presence of methylation is indicative of the presence
of the disease. Methylation of the gene causes a down regulation in
expression which may also be monitored. Related methods and kits are also
described based upon methylation of miRNA encoding genes. The primary
miRNA of interest is 124a miRNA.

Claims:

1. A method of diagnosing a disease in which an miRNA is expressed at a
lower level comprising, in a test sample obtained from a subject,
determining the methylation status of at least one gene encoding the
miRNA in the sample, wherein the presence of methylation is indicative of
the presence of the disease.

2. A method for predicting the likelihood of successful treatment or
resistance to treatment of a disease in which an miRNA is expressed at a
lower level with a DNA demethylating agent and/or a DNA methyltransferase
inhibitor and/or a HDAC inhibitor comprising determining the methylation
status of at least one gene encoding the miRNA in a sample obtained from
a subject, wherein if the gene is methylated the likelihood of successful
treatment is higher than if the gene is unmethylated and wherein if the
gene is unmethylated the likelihood of resistance to treatment is higher
than if the gene is methylated.

3. (canceled)

4. A method of selecting a suitable treatment regimen for a disease in
which an miRNA is expressed at a lower level comprising determining the
methylation status of at least one gene encoding the miRNA in a sample
obtained from a subject, wherein if the gene is methylated a DNA
demethylating agent and/or a DNA methyltransferase inhibitor and/or a
HDAC inhibitor is selected for treatment.

5. The method of claim 1 wherein the methylation status of the gene in the
test sample is compared to that of a control sample.

10. The method according to claim 1 which comprises determining the
methylation status of the gene or genes encoding 124a miRNA.

11. (canceled)

12. The method according to claim 1, wherein the methylation status is
determined using a technique selected from methylation specific PCR,
bisulphite sequencing, microarray techniques, real-time or end point
amplification techniques and COBRA, either alone or in combination.

13. (canceled)

14. The method according to claim 12 wherein the methylation specific PCR
comprises use of primer pairs selected from the primers comprising the
nucleotide sequences set forth as SEQ ID NO: 2 and 3/6 and 7/10 and 11/or
functional derivatives thereof in order to determine if the gene or genes
encoding 124a miRNA is/are methylated.

15. The method according to claim 12 wherein the methylation specific PCR
comprises use of primer pairs selected from the primers comprising the
nucleotide sequences set forth as SEQ ID NO: 4 and 5/8 and 9/12 and 13 or
functional derivatives thereof in order to determine if the gene or genes
encoding 124a miRNA are unmethylated.

16. The method of claim 12 wherein bisulphite sequencing of the gene or
genes encoding 124a miRNA is carried out by using sequencing primer pairs
selected from the primers comprising the nucleotide sequences set forth
as SEQ ID NO: 14 and 15/16 and 17/19 and 19 or functional derivatives
thereof.

17. A method of diagnosing cancer comprising, in a test sample obtained
from a subject, determining the expression levels or activity of 124a
miRNA in the sample, wherein a reduced level of expression or activity is
indicative of the presence of the disease.

18. A method for predicting the likelihood of successful treatment or
resistance to treatment of cancer with a DNA demethylating agent and/or a
DNA methyltransferase inhibitor and/or a HDAC inhibitor comprising
determining the expression levels or activity of 124a miRNA in a test
sample obtained from a subject, wherein a reduced level of expression or
activity is indicative of the likelihood of successful treatment being
higher than if the expression levels or activity are higher and wherein
an increased level of expression or activity is indicative of the
likelihood of successful treatment being lower than if the expression
levels or activity are lower.

19. (canceled)

20. A method of selecting a suitable treatment regimen for cancer
comprising determining the expression levels or activity of 124a miRNA in
a test sample obtained from a subject, wherein if the expression levels
or activity are lower a DNA demethylating agent and/or a DNA
methyltransferase inhibitor and/or a HDAC inhibitor is selected for
treatment.

21. The method of claim 17 wherein the expression levels of the 124a miRNA
in the test sample is compared to that of a control sample.

33. A recombinant construct capable of expressing an miRNA comprising the
nucleotide sequence set forth as SEQ ID NO: 1 or a functional derivative
thereof with the ability to down-regulate CDK6 protein expression.

34. (canceled)

35. A method of treating a condition characterised by methylation of miRNA
comprising administering to a patient in need of treatment a
demethylating agent in an amount effective to restore miRNA function.

36. A method of treating a condition characterised by methylation of miRNA
comprising administering to a patient in need of treatment one or more
miRNAs, wherein the administered miRNAs complement miRNA function
suppressed by methylation in the patient.

37. The method of claim 35 or 36 wherein the condition comprises cancer

42. The method of claim 40 wherein the levels and/or activity of CDK6 are
reduced by removing methylation of one or more genes encoding an miRNA
molecule, which miRNA molecule acts to inhibit expression of CDK6 and/or
by administering to the patient one or more miRNAs, wherein the
administered miRNAs act to reduce the levels and/or activity of CDK6 in
the patient.

43. The method of claim 42 wherein the one or more genes encoding an miRNA
molecule whose methylation is removed comprises the one or more genes
encoding 124a miRNA.

44. The method of claim 42 wherein the one or more miRNA molecules which
is administered comprises 124a miRNA or a functional derivative thereof
with the ability to down-regulate CDK6 protein expression.

45. A pharmaceutical composition comprising an miRNA together with a
suitable carrier/excipient/diluent.

46. The composition of claim 45 wherein the miRNA comprises 124a miRNA or
a functional derivative thereof with the ability to down-regulate CDK6
protein expression.

47. The composition of claim 45 which comprises a recombinant construct or
a host cell or vector.

48. (canceled)

49. (canceled)

50. (canceled)

51. (canceled)

52. A kit for:(a) diagnosing a disease in which an miRNA is expressed at a
lower level; and/or(b) predicting the likelihood of successful treatment
of a disease in which an miRNA is expressed at a lower level and/or the
likelihood of resistance to treatment of a disease in which an miRNA is
expressed at a lower level with a DNA demethylating agent and/or a DNA
methyltransferase inhibitor and/or a HDAC inhibitor, and/or(c) selecting
a suitable treatment regimen for a disease in which an miRNA is expressed
at a lower levelcomprising carrier means containing therein a set of
primers for use in detecting the methylation status of at least one gene
encoding the miRNA or for use in detecting the expression level of an
miRNA which is expressed at a lower level in the disease.

53. The kit according to claim 52 wherein the miRNA is 124a miRNA.

54. The kit according to claim 53 for use in bisulphite sequencing of one
or more genes encoding 124a miRNA comprising at least one sense and
antisense primer pair selected from the primers comprising the nucleotide
sequences set forth as SEQ ID NO 14, 15, 16, 17, 18 and 19, and
functional derivatives thereof which are useful in bisulfite sequencing
of one or more genes encoding 124a miRNA.

55. The kit according to claim 53 comprising at least one sense and
antisense primer pair selected from the primers comprising the nucleotide
sequences set forth as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and
13 and functional derivatives thereof which are useful in MSP for
determining the methylation status of one or more genes encoding 124a
miRNA.

56. (canceled)

57. The kit according to claim 52 comprising a bisulphite reagent.

58. (canceled)

59. (canceled)

60. The kit according to claim 52 for use in RT-PCR comprising at least
one sense and antisense primer pair selected from the primers comprising
the nucleotide sequences set forth as SEQ ID NO: 32 and 33 or SEQ ID NO:
37 and 38 respectively and functional derivatives thereof which are
useful in RT-PCR for determining expression levels of 124a miRNA.

61. (canceled)

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001]This application claims priority to U.S. Provisional Patent
Application No. 60/854,726, filed on Oct. 27, 2006. The contents of that
application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002]The present invention relates to diagnosis and therapy. In
particular, the invention relates to cancer diagnosis and treatment. More
particularly, the invention relates to micro RNA molecules (miRNA),
especially miR-124a, and the role that methylation of genes encoding
miRNAs plays in disease.

BACKGROUND OF THE INVENTION

[0003]MicroRNAs (miRNAs) are short, approximately 22 nucleotide,
non-coding RNAs that are thought to regulate gene expression by
sequence-specific base pairing in the 3' unstranslated regions of the
targets mRNA inducing direct mRNA degradation or translational inhibition
(1,2). miRNA expression patterns can be developmentally regulated,
tissue-specific or steadily expressed in the whole organism (1,2) and are
considered to play important roles in cell proliferation, apoptosis and
differentiation (1,2). In disease, recent studies have shown that miRNA
expression profiles are distinct between normal tissues and the
derived-tumors (3) and between different tumor types (3). Interestingly,
downregulation of subsets of miRNAs is a common finding in many of these
studies (2,3), suggesting that some of these miRNAs may act as putative
tumor suppressor genes.

[0004]This last indication has been studied in more detail for particular
cases, and for example the down-regulated let-7, miR-15/miR-16 and
miR-127 target the oncogenic factors RAS, BCL-2 and BCL-6, respectively
(4-6). One explanation is a failure at the post-transcriptional
regulation of these microRNAs in cancer cells (7). However, additional
mechanisms could also be invoked. In this regard, restoration of
microRNA-127 expression in cancer cells by treatment with a DNA
demethylating agent in combination with a histone deacetylase inhibitor
has been recently reported (6 & 8). However, the gene encoding this micro
RNA was shown to be heavily methylated both in normal cells and in cancer
cells.

DESCRIPTION OF THE INVENTION

[0005]The present invention is based around the discovery of a direct link
between methylation of a gene encoding an miRNA and cancer. In
particular, methylation of the genes encoding miR-124a has been shown to
be functionally linked to a down-regulation of this miRNA in cancer
cells, with a consequential effect on oncogene activity. This link
between methylation status of an miRNA encoding gene and cancer is useful
in a number of diagnostic and therapeutic applications.

[0006]Accordingly, in a first aspect the invention provides a method of
diagnosing a disease in which an miRNA expression profile is altered
comprising, in a test sample obtained from a subject, determining the
methylation status of at least one gene/DNA encoding the miRNA in the
sample, wherein the presence of methylation, in particular
hypermethylation, is indicative of (a positive diagnosis of) the presence
of the disease.

[0007]Preferably, the miRNA is expressed at a lower level in the disease
condition than in normal subjects. Methylation causes down regulation of
miRNA expression from its own promoter and thus can be used as a
diagnostic indicator of the disease.

[0008]"Diagnosis" is defined herein to include monitoring the state and
progression of the disease, checking for recurrence of disease following
treatment and monitoring the success of a particular treatment. The tests
may also have prognostic value, and this is included within the
definition of the term "diagnosis". The prognostic value of the tests may
be used as a marker of potential susceptibility to the disease in
question. Thus patients at risk may be identified before the disease has
a chance to manifest itself in terms of symptoms identifiable in the
patient.

[0009]According to a further aspect, the invention provides a method for
predicting the likelihood of successful treatment of a disease, in which
an miRNA expression profile is altered, with a DNA demethylating agent
and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor
comprising determining the methylation status of at least one gene/DNA
encoding the miRNA in a sample obtained from a subject, wherein if the
gene/DNA is methylated, in particular hypermethylated, the likelihood of
successful treatment is higher than if the gene/DNA is unmethylated or
methylated at a lower level (i.e. not hypermethylated). Preferably, the
miRNA is expressed at a lower level in the disease condition than in
normal subjects.

[0010]The opposite scenario is also envisaged in the present invention.
Thus, in a related aspect, the invention provides a method for predicting
the likelihood of resistance to treatment of a disease in which an miRNA
expression profile is altered with a DNA demethylating agent and/or a DNA
methyltransferase inhibitor and/or a HDAC inhibitor comprising
determining the methylation status of at least one gene/DNA encoding the
miRNA in a sample obtained from a subject, wherein if the gene/DNA is
unmethylated, or methylated at a lower level (i.e. not hypermethylated)
the likelihood of resistance to treatment is higher than if the gene/DNA
is methylated, in particular hypermethylated. Preferably, the miRNA is
expressed at a lower level in the disease condition than in normal
subjects.

[0011]Since methylation of the 124a miRNA genes manifests itself in down
regulation of gene expression, the invention also provides a method of
selecting a suitable treatment regimen for a disease in which an miRNA is
expressed at a lower level (or in which the expression profile is
altered) comprising determining the methylation status of at least one
gene encoding the miRNA in a sample obtained from a subject, wherein if
the gene is methylated, in particular hypermethylated, a DNA
demethylating agent and/or a DNA methyltransferase inhibitor and/or a
HDAC inhibitor is selected for treatment.

[0012]The opposite scenario is also envisaged in the present invention.
Thus, in a related aspect, the detection of an unmethylated, or
methylated at a lower level (i.e. not hypermethylated) miRNA gene,
preferably one or more 124a miRNA genes, indicates that treatment with a
DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a
HDAC inhibitor is contra-indicated. Accordingly, alternative treatments
should be explored.

[0013]In each of the above methods, confirmation of the effect of the DNA
demethylating agent and/or a DNA methyltransferase inhibitor and/or a
HDAC inhibitor may be sought by further contacting the sample with the
DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a
HDAC inhibitor and determining whether the methylation status of the one
or more miRNA genes is altered.

[0014]By "likelihood of successful treatment" is meant the probability
that treatment of the disease using any one or more of the listed
therapeutic agents will be successful.

[0015]"Successful treatment" is defined to include complete recovery and
also significant regression of the disease, and improved survival rates.
Improved alleviation of symptoms may also be considered as "successful
treatment" in the present invention.

[0016]"Resistance" is defined as a reduced probability that treatment of
the disease will be successful using any one of the specified therapeutic
agents and/or that higher doses will be required to achieve a therapeutic
effect.

[0017]By "DNA demethylating agent" is meant any molecule, compound or
otherwise which is capable of reducing, removing or inhibiting
methylation. In particular, methylation of one or more genes encoding a
disease associated miRNA (such as 124a miRNA) is inhibited by the DNA
demethylating agent. This increases expression of the miRNA.

[0018]By "DNA methyltransferase inhibitor" is meant any molecule, compound
or otherwise which is capable of inhibiting DNA methyltransferase
activity and thus acting to up-regulate expression of the miRNA which is
down-regulated in the relevant disease condition. DNA methyltransferases
catalyze transfer of a methyl group to DNA. In particular, DNA
methyltransferases are responsible for methylation of DNA at CpG sites,
which can cause down regulation of important tumour suppressor genes,
such as the genes encoding 124a miRNA. Although not bound by this theory,
inhibiting DNA methyltransferase activity is thought to prevent down
regulation of important tumour suppressor genes through methylation, thus
leading to the effective treatment of diseases such as cancer. Inhibitors
of DNA methyltransferase activity may influence expression of the DNA
methyltransferases or may inhibit the enzymatic activity of the protein
for example.

[0019]"HDAC inhibitor" is defined as any molecule, compound or otherwise
capable of inhibiting histone deacetylase activity and thus acting to
up-regulate expression of the miRNA which is down-regulated in the
relevant disease condition. Histone deacetylases are a class of enzymes
which are responsible for removal of acetyl groups from lysine amino
acids in histones. This removal of an acetyl group means that the
positively charged lysine becomes available for interaction with DNA. The
interaction of histones with DNA generally down regulates gene expression
by blocking access to the DNA of components required for transcription.
Many HDAC inhibitors are in clinical trials and are known to be useful in
treating cancer. Inhibitors of HDAC activity may influence expression of
the HDACs or may inhibit the enzymatic activity of the protein for
example.

[0020]By "methylation status" is meant the level of methylation of
cytosine residues (found in CpG pairs) in the relevant miRNA gene which
are relevant to the regulation of gene expression. Thus, the levels of
methylation of an miRNA gene are determined by any suitable means in
order to reflect whether the gene is likely to be down regulated or not.
Generally, an increase in methylation is associated with a corresponding
decrease in gene expression.

[0021]"Expression levels", unless otherwise stated, are defined to include
levels of mRNA produced by transcription of the appropriate
miRNA-encoding gene. Changes in the level of expression may be measured
directly or indirectly. Indirect measurement may involve determining
expression of genes, at either the mRNA or protein level, whose
expression is modified or at least partially determined by the relevant
miRNA.

[0022]"Hypermethylation" is a well-known term in the art. It is defined as
an increase in the level of methylation above normal levels. Thus, it
relates to aberrant methylation at specific CpG sites in a gene, often in
the promoter region. The terms "methylation" and "hypermethylation" may
be utilised interchangeably herein. Normal levels of methylation may be
defined by comparison to non-diseased cells for example. Methylation and
hypermethylation are generally linked to down regulation of gene
expression. In this invention, methylation and in particular
hypermethylation of specific miRNA genes, such as the 124a miRNA genes,
is indicative of a loss of expression of these important miRNA products
and thus provides a reliable indicator of the disease condition.

[0023]"Suitable treatment regimen" is defined to include the choice of
treatment which is to be made by the individual carrying out the method.
The regimen chosen is one deemed suitable on the basis of the methylation
status and/or expression levels of the relevant miRNA gene and is
selected from a DNA demethylating agent and/or a DNA methyltransferase
inhibitor and/or a HDAC inhibitor according to the invention. Of course,
as would be readily appreciated by the skilled practitioner, the specific
dosage regime may be calculated according to the body surface area of the
patient or the volume of body space to be occupied, dependent on the
particular route of administration to be used. The amount of the
composition actually administered will, however, be determined by a
medical practitioner based on the circumstances pertaining to the
disorder to be treated, such as the severity of the symptoms, the age,
weight and response of the individual.

[0024]All methods of the invention may be carried out with respect to any
gene encoding an miRNA, and whose expression is altered due to
methylation or hypermethylation in a disease condition. Thus, for the
first time herein methylation of miRNA encoding genes has been shown to
be disease associated. Particularly preferred is the 124a miRNA and the
genes encoding this molecule which are shown herein for the first time to
be methylated and thus the miRNA is down-regulated in various cancer
types.

[0025]The terms miR-124a and 124a miRNA are used interchangeably herein.
This particular miRNA molecule is encoded by three separate genes located
at three different chromosomal locations within the human genome. The
approved gene symbol (as per the HUGO gene nomenclature committee, see
www.gene.ucl.ac.uk/nomenclature) for each of the three genes is
MIRN124A1, MIRN124A2 and MIRN124A3 respectively. The corresponding
approved gene names are microRNA 124a-1, microRNA 124a-2 and microRNA
124a-3 respectively. The respective chromosomal locations for these genes
is 8p23.1, 8q12.3 and 20q13.33 respectively. Aliases include
hsa-mir-124a-1, hsa-mir-124a-2 and hsa-mir-124a-3 respectively. Thus,
reference to "one or more genes encoding 124a miRNA" is intended to cover
these genes. Of course, as appropriate, the skilled person would
appreciate that functionally relevant variants of each of the gene
sequences may also be detected according to the methods of the invention.
For example, the methylation status of a number of splice variants may be
determined according to the methods of the invention. Variant sequences
preferably have at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% nucleotide sequence identity with the nucleotide sequences
in the database entries. Computer programs for determining percentage
nucleotide sequence identity are available in the art, including the
Basic Local Alignment Search Tool (BLAST) available from the National
Center for Biotechnology Information. The miR-124a/124a miRNA molecule
has the following nucleotide sequence:

TABLE-US-00001
ACCGUAAGUGGCGCACGGAAUU. (SEQ ID NO: 1)

[0026]All of these methods are most preferably in vitro methods carried
out on an isolated sample. In one embodiment, the methods may also
include the step of obtaining the sample.

[0027]In a most preferred embodiment, the subject is a human subject.
Generally the subject may be a patient wherein a potential disease, in
particular cancer, has been identified and the method may be used to
determine if indeed there is a potentially dangerous condition and also
to guide treatment depending upon the methylation status of the one or
more miRNA encoding gene(s).

[0028]The test sample is generally any sample taken preferably from the
subject under test in which the methylation status of the at least one
miRNA encoding genes is reflective of the disease status. The sample may
comprise a tissue sample taken from the subject which is suspected of
being a tumor, wherein the disease is preferably cancer. The tissue
chosen may be determined by the type of cancer which is suspected or is
to be treated. Preferred cancer types according to the methods of the
invention are discussed in more detail below and the skilled person would
immediately appreciate which type of sample would be appropriate
depending upon the cancer concerned (for example suitable colon samples
for diagnosing colon cancer).

[0029]However, any other suitable test sample in which methylation status
of at least one gene encoding an miRNA, most preferably the 124a miRNA
gene, can be determined to indicate the presence of disease or the
likelihood of successful treatment of the disease with the specified
agents or indeed to select a suitable treatment for the disease are
included within the scope of the invention. Test samples for diagnostic,
prognostic, or personalised medicinal uses may be obtained from surgical
samples, such as biopsies or fine needle aspirates, from paraffin
embedded tissues or from a body fluid for example. Non-limiting examples
of samples which may be used, as appropriate, in the present invention
include whole blood, serum, plasma, urine, chyle, stool, ejaculate,
sputum, nipple aspirate and saliva.

[0030]All of these aspects of the invention rely upon determining the
methylation status of at least one gene encoding an miRNA, preferably
124a miRNA.

[0031]The methylation status is determined for the promoter region of the
gene or genes encoding the miRNA. The methylation status may, however,
(also or alternatively) be determined in introns and exons as
appropriate. Thus, methylation of the gene, and often the promoter of the
gene, encoding the miRNA causes down regulation of expression of the
miRNA which is associated with the disease. Preferably, the methylation
status of at least one defined CpG island, which may be found (at least
partially) in the promoter region, of the gene encoding the
diagnostically useful miRNA is determined. CpG islands in an miRNA
encoding gene can be readily determined utilising the CpG Island Search
Program for example (see http://cpgislands.usc.edu/ and Takai D, Jones P
A. The CpG island searcher: a new WWW resource. In Silico Biol. 3,
235-240 (2003)).

[0032]Most preferably, the methods of the invention include determining
the methylation status of at least one of the genes encoding 124a miRNA
(SEQ ID NO: 1 ACCGUAAGUGGCGCACGGAAUU). As discussed in greater detail in
the experimental section below, methylation of the genes encoding 124a
miRNA has been shown for the first time to influence expression levels of
the miRNA with functional consequences in terms of recognised oncogene
activity. 124a miRNA is encoded by genes found at three separate loci.
Thus, the methods of the invention may be carried out to determine
methylation of the gene at any one, two or all of these loci. Accordingly
methylation levels at the genomic loci 8p23.1/8q12.3/20q13.33 may be
determined in one embodiment. As discussed above, preferably methylation
status of the CpG islands identified in these genes is determined.

[0033]It is noted that the methylation status of additional genes, in
particular tumour suppressor genes, may also be determined in order to
supplement the methods of the invention.

[0034]In one preferred embodiment of these aspects of the invention the
methylation status of the at least one gene encoding suitable miRNA, in
particular the at least one genes encoding 124a miRNA, (or portion
thereof, especially the CpG islands) is determined using methylation
specific PCR (MSP). However, any suitable technique may be utilised to
determine the methylation status of the at least one gene encoding an
miRNA, which is most preferably 124a miRNA. Various methylation assay
procedures are known in the art, and can be used in conjunction with the
present invention. These assays rely upon two distinct approaches:
bisulphite conversion based approaches and non-bisulphite based
approaches. Non-bisulphite based methods for analysis of DNA methylation
rely on the inability of methylation-sensitive enzymes to cleave
methylation cytosines in their restriction. The bisulphite conversion
relies on treatment of DNA samples with sodium bisulphite which converts
unmethylated cytosine to uracil, while methylated cytosines are
maintained (Furuichi et al., 1970). This conversion results in a change
in the sequence of the original DNA.

[0036]Additional methods for the identification of methylated CpG
dinucleotides utilize the ability of the methyl binding domain (MBD) of
the MeCP2 protein to selectively bind to methylated DNA sequences (Cross
et al, 1994; Shiraishi et al, 1999). Alternatively, the MBD may be
obtained from MBP, MBP2, MBP4 or poly-MBD (Jorgensen et al., 2006). In
one method, restriction exonuclease digested genomic DNA is loaded onto
expressed His-tagged methyl-CpG binding domain that is immobilized to a
solid matrix and used for preparative column chromatography to isolate
highly methylated DNA sequences. Such methylated DNA enrichment-step may
supplement the methods of the invention. Several other methods for
detecting methylated CpG islands are well known in the art and include
amongst others methylated-CpG island recovery assay (MIRA). Any of these
methods may be employed in the present invention where desired.

[0037]The MSP technique will be familiar to one of skill in the art. In
the MSP approach, DNA may be amplified using primer pairs designed to
distinguish methylated from unmethylated DNA by taking advantage of
sequence differences as a result of sodium-bisulfite treatment (Herman J
G, Graff J R, Myohanen S, Nelkin B D, Baylin S B. Methylation-specific
PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl
Acad Sci USA 1996; 93(18):9821-9826 and see WO 97/46705, incorporated
herein by reference). After sodium-bisulfite treatment unmethylated
cytosines are converted to uracil whereas methylated cytosines remain
unconverted.

[0038]A specific example of the MSP technique is designated real-time
quantitative MSP (QMSP), which permits reliable quantification of
methylated DNA in real time. These methods are generally based on the
continuous optical monitoring of an amplification procedure and utilise
fluorescently labelled reagents whose incorporation in a product can be
quantified and whose quantification is indicative of copy number of that
sequence in the template. One such reagent is a fluorescent dye, called
SYBR Green I that preferentially binds double-stranded DNA and whose
fluorescence is greatly enhanced by binding of double-stranded DNA.
Alternatively, labelled primers and/or labelled probes can be used. They
represent a specific application of the well known and commercially
available real-time amplification techniques such as hydrolytic probes
(TAQMAN®), hairpin probes (MOLECULAR BEACONS®), hairpin primers
(AMPLIFLUOR®), hairpin probes integrated into primers
(SCORPION®)), oligonucleotide blockers and primers incorporating
complementary sequences of DNAzymes (DzyNA®), specific interaction
between two modified nucleotides (Plexor®) etc as described in more
detail herein. Often, these real-time methods are used with the
polymerase chain reaction (PCR). In Heavymethyl, the priming is
methylation specific, but non-extendable oligonucleotide blockers provide
this specificity instead of the primers themselves. The blockers bind to
bisulfite-treated DNA in a methylation-specific manner, and their binding
sites overlap the primer binding sites. When the blocker is bound, the
primer cannot bind and therefore the amplicon is not generated.
Heavymethyl can be used in combination with real-time detection in the
methods of the invention.

[0040]Real-Time PCR detects the accumulation of amplicon during the
reaction. Real-time methods do not need to be utilised, however. Many
applications do not require quantification and Real-Time PCR is used
principally as a tool to obtain convenient results presentation and
storage, and at the same time to avoid post-PCR handling. Analyses can be
performed only to know if the target DNA is present in the sample or not.
End point verification is carried out after the amplification reaction
has finished. This knowledge can be used in a medical diagnostic
laboratory to detect a predisposition to, or the incidence of, cancer in
a patient. In the majority of such cases, the quantification of DNA
template is not very important. Amplification products may simply be run
on a suitable gel, such as an agarose gel, to determine if the expected
sized products are present. This may involve use of ethidium bromide
staining and visualisation of the DNA bands under a UV illuminator for
example. Alternatively, fluorescence or energy transfer can be measured
to determine the presence of the methylated DNA. The end-point PCR
fluorescence detection technique can use the same approaches as widely
used for Real Time PCR: TaqMan assay, Molecular Beacons, Scorpion,
Amplifluor etc as discussed herein. For example, <<Gene>>
detector allows the measurement of fluorescence directly in PCR tubes.

[0041]In real-time embodiments, quantitation may be on an absolute basis,
or may be relative to a constitutively methylated DNA standard, or may be
relative to an unmethylated DNA standard. Methylation status may be
determined by using the ratio between the signal of the marker under
investigation and the signal of a reference gene where methylation status
is known (such as β-actin for example), or by using the ratio
between the methylated marker and the sum of the methylated and the
non-methylated marker. Alternatively, absolute copy number of the
methylated marker gene can be determined. Suitable reference genes for
the present invention include beta-actin, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), ribosomal RNA genes such as 18S ribosomal RNA and
RNA polymerase II gene (Radonic A. et al., Biochem Biophys Res Commun.
2004 Jan. 23; 313(4):856-62). In a particularly preferred embodiment, the
reference gene is beta-actin.

[0042]In one embodiment, each clinical sample is measured in duplicate and
for both Ct values (cycles at which the amplification curves crossed the
threshold value, set automatically by the relevant software) copy numbers
are calculated. The average of both copy numbers (for each gene) is used
for the result classification. To quantify the final results for each
sample two standard curves are used, one for either the reference gene
(β-actin or the non-methylated marker) and one for the methylated
version of the marker. The results of all clinical samples (when m-Gene
was detectable) are expressed as 1000 times the ratio of "copies
m-Gene"/"copies β-actin" or "copies m-Gene"/"copies u-Gene+m-Gene"
and then classified accordingly (methylated, non-methylated or invalid)
(u=unmethylated; m=methylated).

[0043]In one embodiment, primers useful in MSP carried out on the promoter
region of the respective 124a miRNA genes are provided (MIRN124A1,
MIRN124A2 and MIRN124A3). These primers comprise, consist essentially of
or consist of any suitable combinations selected from the following
sequences:

[0044]Within the scope of the invention are functional derivatives of
these primers. The primers may contain minor variations, such as single
nucleotide substitutions, small additions and deletions, provided that
their ability to act as MSP primers is not compromised. Thus, variant
primers must retain the ability to distinguish between methylated and
unmethylated nucleic acid molecules, in particular genes encoding 124a
miRNA, following bisulphite treatment. Thus, the primers may be between
10 and 50 nucleotides in length, preferably between 12 and 40 nucleotides
and most preferably between 15 and 30 nucleotides. Other variants of
these sequences may be utilised in the present invention. In particular,
additional sequence specific flanking sequences may be added, for example
to improve binding specificity, as required. Variant sequences preferably
have at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% nucleotide sequence identity with the primers set forth herein. The
primers may be adapted to allow real-time or end point fluorescence
detection. For example, they may incorporate hairpin probe structures.
The primers and/or hairpin structures may incorporate synthetic
nucleotide analogues as appropriate or may be RNA or PNA based for
example, or mixtures thereof. Similarly alternative fluorescent donor and
acceptor moieties/FRET pairs may be utilised as appropriate. In addition
to being labelled with the fluorescent donor and acceptor moieties, the
primers may include modified oligonucleotides and other appending groups
and labels provided that the functionality as a primer in the methods of
the invention is not compromised. Similarly alternative fluorescent donor
and acceptor moieties/FRET pairs may be utilised as appropriate.
Molecules that are commonly used in real-time and end point detection
techniques include fluorescein, 5-carboxyfluorescein (FAM),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), rhodamine,
6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine
(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)
benzoic acid (DABCYL), and 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic
acid (EDANS). Whether a fluorophore is a donor or an acceptor is defined
by its excitation and emission spectra, and the fluorophore with which it
is paired. For example, FAM is most efficiently excited by light with a
wavelength of 488 nm, and emits light with a spectrum of 500 to 650 nm,
and an emission maximum of 525 nm. FAM is a suitable donor fluorophore
for use with JOE, TAMRA, and ROX (all of which have their excitation
maximum at 514 nm).

[0046]In one specific embodiment, said donor moiety is fluorescein or a
derivative thereof, and said acceptor moiety is DABCYL. Preferably, the
fluorescein derivative comprises, consists essentially of or consists of
6-carboxy fluorescein.

[0047]For all aspects and embodiments of the invention, the primers and in
particular the stem loop/hairpin structures, may be labelled with donor
and acceptor moieties during chemical synthesis of the primers or the
label may be attached following synthesis using any suitable method. Many
such methods are available and well characterised in the art.

[0048]In a further embodiment, bisulphite sequencing is utilised in order
to determine the methylation status of the at least one gene encoding an
miRNA, which is most preferably 124a miRNA. Primers may be designed for
use in sequencing through the important CpG islands in the at least one
miRNA gene. Thus, primers may be designed in both the sense and antisense
orientation to direct sequencing across the promoter region of the
relevant gene. In one embodiment, in which the at least one genes
encoding 124a miRNA are sequenced, bisulphite sequencing may be carried
out by using sequencing primers which comprise, consist essentially of or
consist of the following sequences, and which may be used in isolation or
in combination to sequence both strands:

[0049]These sequencing primers form a further aspect of the invention.
Within the scope of the invention are functional derivatives of these
primers. The primers may contain minor variations, such as single
nucleotide substitutions, small additions and deletions, provided that
their ability to act as primers in bisulphite sequencing is not
compromised. Thus, variant primers must retain the ability to distinguish
between methylated and unmethylated nucleic acid molecules, in particular
genes encoding 124a miRNA, when used in bisulphite sequencing. The
variants discussed in respect of MSP primers above applies mutatis
mutandis to this aspect of the invention.

[0050]Other nucleic acid amplification techniques, in addition to PCR
(which includes real-time versions thereof and variants such as nested
PCR), may also be utilised, as appropriate, to detect the methylation
status of the at least one gene encoding an miRNA, which is most
preferably 124a miRNA. Such amplification techniques are well known in
the art, and include methods such as NASBA (Compton, 1991, 3SR (Fahy et
al., 1991) and Transcription Mediated Amplification (TMA). Other suitable
amplification methods include the ligase chain reaction (LCR) (Barringer
et al, 1990), selective amplification of target polynucleotide sequences
(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain
reaction (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain
reaction (WO 90/06995) and nick displacement amplification (WO
2004/067726). This list is not intended to be exhaustive; any nucleic
acid amplification technique may be used provided the appropriate nucleic
acid product is specifically amplified. Thus, these amplification
techniques may be tied in to MSP and/or bisulphite sequencing techniques
for example.

[0051]Sequence variation that reflects the methylation status at CpG
dinucleotides in the original genomic DNA offers two approaches to primer
design. Firstly, primers may be designed that themselves do not cover any
potential sites of DNA methylation. Sequence variation at sites of
differential methylation are located between the two primers. Such
primers are used in bisulphite genomic sequencing, COBRA and Ms-SnuPE for
example. Secondly, primers may be designed that anneal specifically with
either the methylated or unmethylated version of the converted sequence.
If there is a sufficient region of complementarity, e.g., 12, 15, 18, or
20 nucleotides, to the target, then the primer may also contain
additional nucleotide residues that do not interfere with hybridization
but may be useful for other manipulations. Examples of such other
residues may be sites for restriction endonuclease cleavage, for ligand
binding or for factor binding or linkers or repeats. The oligonucleotide
primers may or may not be such that they are specific for modified
methylated residues.

[0052]One way to distinguish between modified and unmodified DNA is to
hybridize oligonucleotide primers which specifically bind to one form or
the other of the DNA. After hybridization, an amplification reaction can
be performed and amplification products assayed. The presence of an
amplification product indicates that a sample hybridized to the primer.
The specificity of the primer indicates whether the DNA had been modified
or not, which in turn indicates whether the DNA had been methylated or
not.

[0053]Another related means for distinguishing between modified and
unmodified DNA is to use oligonucleotide probes which may also be
specific for certain products. Such probes may be hybridized directly to
modified DNA or to amplification products of modified DNA.
Oligonucleotide probes can be labelled using any detection system known
in the art. These include but are not limited to fluorescent moieties,
radioisotope labelled moieties, bioluminescent moieties, luminescent
moieties, chemiluminescent moieties, enzymes, substrates, receptors, or
ligands.

[0054]In the MSP technique, amplification is achieved with the use of
primers specific for the sequence of the gene whose methylation status is
to be assessed. In order to provide specificity for the nucleic acid
molecules, primer binding sites corresponding to a suitable region of the
sequence may be selected. The skilled reader will appreciate that the
nucleic acid molecules may also include sequences other than primer
binding sites which are required for detection of the methylation status
of the gene, for example RNA Polymerase binding sites or promoter
sequences may be required for isothermal amplification technologies, such
as NASBA, 3SR and TMA.

[0055]TMA (Gen-probe Inc.) is an RNA transcription amplification system
using two enzymes to drive the reaction, namely RNA polymerase and
reverse transcriptase. The TMA reaction is isothermal and can amplify
either DNA or RNA to produce RNA amplified end products. TMA may be
combined with Gen-probe's Hybridization Protection Assay (HPA) detection
technique to allow detection of products in a single tube. Such single
tube detection is a preferred method for carrying out the invention.

[0056]As mentioned above, in a preferred embodiment, the methylation
status of the at least one gene encoding an miRNA, which is most
preferably 124a miRNA, is determined by methylation specific PCR,
preferably real-time or end-point methylation specific PCR.

[0057]Since the promoters of the genes encoding 124a miRNA appear to be
almost completely unmethylated in normal tissues, and highly methylated
in cancer, it is clear that the methods of the invention are particularly
useful, since the detection of methylation in this region is readily
observable as being significant in terms of a cancer diagnosis and indeed
in diagnosis of any other disease in which 124a miRNA is involved and
also in selecting suitable treatment regimens and for determining the
likelihood of successful treatment or resistance to treatment with
certain anti-cancer agents. Likewise the detection of unmethylated genes
encoding 124a miRNA is also of relevance.

[0058]However, when determining methylation status, it may still be
beneficial to include suitable controls in order to ensure the method
chosen to assess this parameter is working correctly and reliably. For
example, suitable controls may include assessing the methylation status
of a gene known to be methylated. This experiment acts as a positive
control to help to ensure that false negative results are not obtained
(i.e. a conclusion of a lack of methylation is made even though the at
least one gene encoding an miRNA, which is most preferably 124a miRNA
may, in fact, be methylated). The gene may be one which is known to be
methylated in the sample under investigation or it may have been
artificially methylated, for example by using a suitable
methyltransferase enzyme, such as Sssl methyltransferase. In one
embodiment, the at least one gene encoding an miRNA, which is most
preferably 124a miRNA, may be assessed in normal cells such as colon
cells, following treatment with Sssl methyltransferase, as a positive
control. Alternatively, the positive control may be a suitable diseased
sample in which the gene is known to be methylated.

[0059]Additionally or alternatively, suitable negative controls may be
employed with the methods of the invention. Here, suitable controls may
include assessing the methylation status of a gene known to be
unmethylated. This experiment acts as a negative control and helps to
ensure that false positive results are not obtained (i.e. a conclusion of
methylation is made even though the at least one gene encoding an miRNA,
which is most preferably 124a miRNA may, in fact, be unmethylated). The
gene may be one which is known to be unmethylated in the sample under
investigation or it may have been artificially demethylated, for example
by using a suitable DNA methyltransferase inhibitor, such as those
discussed in more detail herein. In one embodiment, the at least one gene
encoding an miRNA, which is most preferably 124a miRNA, may be assessed
in normal cells such as colon cells as a negative control, since it has
been shown for the first time herein that the 124a miRNA gene is almost
completely unmethylated in normal tissues.

[0060]The level of methylation of at least one gene encoding an miRNA,
which is most preferably 124a miRNA may, as necessary, be measured in
order to determine if it is statistically significant in the sample. This
helps to provide a reliable test for the methods of the invention. Any
method for determining whether the levels of methylation of at least one
gene encoding an miRNA, which is most preferably 124a miRNA is
significant may be utilised. Such methods are well known in the art and
routinely employed. For example, statistical analyses may be performed
using an analysis of variance test. Typical P values for use in such a
method would be P values of <0.05 or 0.01 or 0.001 when determining
whether the relative levels of methylation are statistically significant.
A change in methylation, as compared to a suitable negative control as
defined above, may be deemed significant if there is at least a 10%
increase in methylation for example. The test may be made more selective
by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or 50%, for
example, in order to be considered statistically significant.

[0061]In a particularly preferred embodiment of the invention, the disease
which is associated with methylation of at least one gene encoding an
miRNA, which is most preferably 124a miRNA is cancer. Preferably, the
disease or cancer is not blastoma, in particular neuroblastoma, or
sarcoma. In a particularly preferred embodiment of the invention, the
cancer is selected from colon cancer, breast cancer, lung cancer,
leukaemia and lymphoma. In all of these disease conditions, methylation
of the gene encoding 124a miRNA and consequential reduced expression of
124a miRNA has been shown to be diagnostically relevant. A most preferred
disease condition in which methylation of miRNA genes is relevant is
colon cancer.

Expression Level Methods

[0062]The 124a miRNA is the first miRNA shown to be differentially
methylated between a disease state and a non-disease state. In
particular, loss of 124a miRNA expression has been shown for the first
time herein to be linked to the incidence of cancer.

[0063]Accordingly, in a further aspect, the invention provides a method of
diagnosing a disease associated with a loss of 124a miRNA expression
comprising, in a test sample obtained from a subject, determining the
expression levels or activity of 124a miRNA in the sample, wherein a
reduced level of expression or activity is indicative of the presence of
the disease.

[0064]In a related aspect, the invention also provides a method for
predicting the likelihood of successful treatment of a disease associated
with loss of 124a miRNA expression with a DNA demethylating agent and/or
a DNA methyltransferase inhibitor and/or a HDAC inhibitor comprising
determining the expression levels or activity of 124a miRNA in a test
sample obtained from a subject, wherein a reduced level of expression or
activity is indicative of the likelihood of successful treatment being
higher than if the expression levels or activity are higher.

[0065]The opposite scenario is also envisaged in the present invention.
Thus, in a related aspect, the invention provides a method for predicting
the likelihood of resistance to treatment of a disease associated with
loss of 124a miRNA expression with a DNA demethylating agent and/or a DNA
methyltransferase inhibitor and/or a HDAC inhibitor comprising
determining the expression levels or activity of 124a miRNA in a test
sample obtained from a subject, wherein an increased level of expression
or activity is indicative of the likelihood of successful treatment being
lower than if the expression levels or activity are lower.

[0066]Furthermore, the invention also relates to a method of selecting a
suitable treatment regimen for a disease associated with loss of 124a
miRNA expression comprising determining the expression levels or activity
of 124a miRNA in a test sample obtained from a subject, wherein if the
expression levels or activity are lower/reduced a DNA demethylating agent
and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor is
selected for treatment.

[0067]The opposite scenario is also envisaged in the present invention.
Thus, in a related aspect if the expression levels or activity of 124a
miRNA are higher/increased, treatment using a DNA demethylating agent
and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor is
contra-indicated.

[0068]Confirmation of the effect of the DNA demethylating agent and/or a
DNA methyltransferase inhibitor and/or a HDAC inhibitor may be sought by
further contacting the sample with the DNA demethylating agent and/or a
DNA methyltransferase inhibitor and/or a HDAC inhibitor and determining
whether the expression level of 124a miRNA is altered.

[0069]These methods may be combined with the methods described above
including determining the methylation status of miRNA encoding genes, as
appropriate.

[0070]For the avoidance of doubt, it is confirmed that all relevant
definitions and embodiments provided above in respect of methods
including determination of the methylation status of miRNA encoding genes
apply mutatis mutandis to the aspects of the invention in which
expression of 124a miRNA is monitored.

[0071]The level of expression of 124a miRNA may, as necessary, be measured
in order to determine if it is statistically significant in the sample.
This helps to provide a reliable test for the methods of the invention.
Any method for determining whether the expression level of 124a miRNA is
significantly reduced may be utilised. Such methods are well known in the
art and routinely employed. For example, statistical analyses may be
performed using an analysis of variance test. Typical P values for use in
such a method would be P values of <0.05 or 0.01 or 0.001 when
determining whether the relative expression or activity is statistically
significant. A change in expression may be deemed significant if there is
at least a 10% decrease for example. The test may be made more selective
by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or 50%, for
example, in order to be considered statistically significant.

[0072]In a preferred embodiment, the decreased level of expression of 124a
miRNA is determined with reference to a control sample. This control
sample is preferably taken from normal (i.e. non tumorigenic) tissue in
the subject, where expression of 124a miRNA is normal. Additionally or
alternatively control samples may also be utilised in which there is
known to be a lack of expression of 124a miRNA.

[0073]Suitable additional controls may also be included to ensure that the
test is working properly, such as measuring levels of expression or
activity of a suitable reference gene in both test and control samples.

[0074]In a most preferred embodiment, the subject is a human subject.
Generally, the subject will be a patient wherein cancer is suspected or a
potential cancer has been identified and the method may be used to
determine if indeed there is a cancer present. The methods of the
invention may be used in conjunction with known methods for detecting
cancer.

[0075]A decreased or abolished level of 124a miRNA expression, caused by
methylation of the gene, results in lower levels of functional 124a miRNA
and this is indicative of cancer and/or likelihood of successful
treatment and/or directs the course of treatment. Assessment of
expression levels of 124a miRNA may be monitored at the primary
transcript, precursor or mature miRNA level. Thus, miRNA genes are
generally transcribed in the nucleus by RNA polymerase II to form long
primary transcripts (pri-miRNAs), which are capped with
7-methyl-guanosine and are also polyadenylated. Pri-miRNAs are then
processed into approximately 60 nucleotide precursor miRNAs (pre-miRNAs)
which may form imperfect stem-loop structures by virtue of the activity
of Drosha (a nuclear RNase III enzyme) and its co-factor Pasha.
Pre-miRNAs are transported into the cytoplasm and subsequently cleaved by
the cytoplasmic RNase III enzyme Dicer into mature miRNAs.

[0076]Suitable methods for determining expression of 124a miRNA at the RNA
level are well known in the art. Methods employing nucleic acid probe
hybridization to the relevant 124a miRNA transcript may be employed for
measuring the presence and/or level of 124a miRNA. Such methods include
use of nucleic acid probe arrays (microarray technology) and Northern
blots. Advances in genomic technologies now permit the simultaneous
analysis of thousands of genes, although many are based on the same
concept of specific probe-target hybridization.

[0077]Sequencing-based methods are an alternative. These methods started
with the use of expressed sequence tags (ESTs), and now include methods
based on short tags, such as serial analysis of gene expression (SAGE)
and massively parallel signature sequencing (MPSS). Differential display
techniques provide yet another means of analyzing gene expression; this
family of techniques is based on random amplification of cDNA fragments
generated by restriction digestion, and bands that differ between two
tissues identify cDNAs of interest.

[0078]In one embodiment, the levels of 124a miRNA expression are
determined using reverse transcriptase polymerase chain reaction
(RT-PCR). RT-PCR is a well known technique in the art which relies upon
the enzyme reverse transcriptase to reverse transcribe mRNA to form cDNA,
which can then be amplified in a standard PCR reaction. Protocols and
kits for carrying out RT-PCR are extremely well known to those of skill
in the art and are commercially available.

[0079]In a specific embodiment, RT-PCR determination of miRNA expression
includes use of primers comprising, consisting essentially of or
consisting of the following nucleotide sequences:

[0080]Functional derivatives of these sequences are also envisaged. The
primers may contain minor variations, such as single nucleotide
substitutions, small additions and deletions, provided that their ability
to act as RT-PCR primers is not compromised. Thus, variant primers must
retain the ability to allow determination of expression of 124a miRNA
through RT-PCR techniques. Thus, the primers may be between 10 and 50
nucleotides in length, preferably between 12 and 40 nucleotides and most
preferably between 15 and 30 nucleotides.

[0081]In a preferred embodiment, the RT-PCR is carried out in real time
and in a quantitative manner. Real time quantitative RT-PCR has been
thoroughly described in the literature (see Gibson et al for an early
example of the technique) and a variety of techniques are possible. End
point detection may also be utilised as desired, as discussed herein.
Examples include use of hydrolytic probes (TAQMAN®), hairpin probes
(MOLECULAR BEACONS®), hairpin primers (AMPLIFLUOR®), hairpin
probes integrated into primers (SCORPION®), oligonucleotide blockers
and primers incorporating complementary sequences of DNAzymes
(DzyNA®), specific interaction between two modified nucleotides
(Plexor®) etc as described in more detail herein. Often, these
real-time methods are used with the polymerase chain reaction (PCR). All
of these systems are commercially available and well characterised, and
may allow multiplexing (that is, the determination of expression of
multiple genes in a single sample).

[0082]These techniques produce a fluorescent read-out that can be
continuously monitored. Real-time techniques are advantageous because
they keep the reaction in a "single tube". This means there is no need
for downstream analysis in order to obtain results, leading to more
rapidly obtained results. Furthermore, keeping the reaction in a "single
tube" environment reduces the risk of cross contamination and allows a
quantitative output from the methods of the invention. This may be
particularly important in the clinical setting of the present invention.

[0083]In one specific embodiment, real-time RT-PCR is carried out using
SYBR Green as the detection reagent. This reagent is commercially
available as a PCR master mix from Applied Biosystems. In one specific
embodiment, real time RT-PCR includes use of an antisense oligonucleotide
comprising, consisting essentially of or consisting of the nucleotide
sequence GCGAGCACAGAATTAATACGACTC (SEQ ID NO: 37), optionally together
with a sense primer comprising, consisting essentially of or consisting
of the nucleotide sequence TTA AGG CAC GCG GTG MT GCC A (miR-124a) (SEQ
ID NO: 38). Again, functional derivatives which retain function in the
real-time RT-PCR assay are envisaged within the scope of the invention
(as defined above).

[0084]It should be noted that whilst PCR is a preferred amplification
method, to include variants on the basic technique such as nested PCR,
equivalents may also be included within the scope of the invention.
Examples include isothermal amplification techniques such as NASBA, 3SR,
TMA and triamplification, all of which are well known in the art and
commercially available. Other suitable amplification methods include the
ligase chain reaction (LCR) (Barringer et al, 1990), selective
amplification of target polynucleotide sequences (U.S. Pat. No.
6,410,276), consensus sequence primed polymerase chain reaction (U.S.
Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction
(WO90/06995) and nick displacement amplification (WO2004/067726).

[0085]In one embodiment, the methods of the invention are carried out by
determining protein expression of CDK6 as an indication of the
methylation status/expression level of the miRNA. Thus, methylation of
the genes encoding 124a miRNA has been shown herein to cause down
regulation of expression of 124a miRNA and this leads to, as a direct
functional consequence, an increase in the translation of the oncogene
CDK6. This gene was identified and confirmed as a target of 124a miRNA
herein for the first time, as described in further detail in the
experimental section below. In a preferred embodiment, high levels of
protein expression of CDK6 is observed in the sample in order to conclude
a diagnosis of cancer, or to make a decision on the best course of
treatment in accordance with the other methods of the invention. High
levels of expression are indicative of methylation of 124a miRNA and thus
reduced levels of 124a miRNA.

[0086]Relative levels of expression may be determined against suitable
controls, as discussed herein in further detail. Levels of protein
expression may be determined by a number of techniques, as are well known
to one of skill in the art. Examples include western blots,
immunohistochemical staining and immunolocalization, immunofluorescene,
enzyme-linked immunosorbent assay (ELISA), immunoprecipitation assays,
agglutination reactions, radioimmunoassay, flow cytometry and equilibrium
dialysis. These methods generally depend upon a reagent specific for
identification of CDK6. The reagent is preferably an antibody and may
comprise monoclonal or polyclonal antibodies. Fragments and derivatized
(such as humanized) antibodies may also be utilised, to include without
limitation Fab fragments, ScFv, single domain antibodies, nanobodies,
heavy chain antibodies etc which retain specific CDK6 binding function.
Any detection method may be employed in accordance with the invention.
The nature of the reagent is not limited except that it must be capable
of specifically identifying CDK6. Additional examples include lectins,
receptors and nucleic acid based reagents (DNA/RNA etc.) Of course, in
the case of a positive diagnosis of cancer, there will be high levels of
CDK6. This method is favourable, since a positive result is investigated
rather than a negative one. The level of expression of CDK6 may, as
necessary, be measured in order to determine if it is statistically
significant in the sample. This helps to provide a reliable test for the
methods of the invention. Any method for determining whether the
expression level of CDK6 is significantly increased may be utilised. Such
methods are well known in the art and routinely employed. For example,
statistical analyses may be performed using an analysis of variance test.
Typical P values for use in such a method would be P values of <0.05
or 0.01 or 0.001 when determining whether the relative expression or
activity is statistically significant. A change in expression may be
deemed significant if there is at least a 10% increase for example. The
test may be made more selective by making the change at least 15%, 20%,
25%, 30%, 35%, 40% or 50%, for example, in order to be considered
statistically significant.

[0087]Suitable controls are described above and that discussion applies
here mutatis mutandis.

[0088]Also shown herein is the effect of CDK6 kinase activity on
retinoblastoma (Rb) phosphorylation. Rb is a tumour suppressor gene and
represents a target of CDK6 activity. Specifically, measuring levels of
phosphorylation of Rb, in particular at residues 807 and 811 (based upon
the wild type amino acid sequence) provides an indication of the activity
of CDK6 which in turn may provide an insight into the methylation status
of the genes encoding miR-124a and expression levels of 124a miRNA. Thus,
assessing phosphorylation of Rb can also be useful in the present
invention. Methods of determining levels of protein phosphorylation are
known in the art and include use of detection reagents such as antibodies
(including derivatives that retain specific binding affinity) specific
for the phosphorylated form of the protein. Examples include use of
Western Blots, Immunohistochemistry etc. The discussions of determining
the significance in changes to phosphorylation levels, including
comparison with suitable controls, may be derived with reference to the
more detailed discussion above, which applies here mutatis mutandis.

[0089]In similar fashion, the description of preferred disease types,
sample types, subjects etc. above in respect of "methylation" related
aspects of the invention apply mutatis mutandis to the "expression"
aspects of the invention. Thus preferably, the cancer is selected from
colon cancer, breast cancer, lung cancer, leukaemia and lymphoma. The
cancer is most preferably colon cancer. Also, as aforementioned, the 124a
miRNA may be one encoded by the gene or genes found the genomic loci
8p23.1 (124a1 miRNA)/8q12.3 (124a2 miRNA)/20q13.33 (124a3 miRNA).

[0090]In further embodiments of both the "methylation" and "expression"
aspects of the invention, which may be additional to the basic methods or
utilised as alternatives, acetylation of histones H3/H4 is used as an
indication of the methylation status of the at least one gene encoding
expression level of the miRNA. As is shown in the experimental section
below, acetylation of both histones H3 and H4 is linked to demethylation
of 124a miRNA and a consequential up-regulation in expression. In cancer
cells, 124a miRNA is methylated leading to lower levels of 124a miRNA and
this is linked to an absence of histone H3 and H4 acetylation in the CpG
island of the 124a miRNA gene.

[0091]In an additional or alternative embodiment, the methods of the
invention incorporate determining the methylation status of histone H3 as
an indication of the methylation status of the gene encoding/expression
level of the miRNA. As is shown in the experimental section below,
methylation of histone H3 is linked to methylation of 124a miRNA encoding
genes and a consequential down-regulation in 124a miRNA expression in
cancer cells. Removal of methylation of the one or more genes encoding
124a miRNA leading to increased levels of 124a miRNA is linked to a loss
of histone H3 methylation. In a specific embodiment, trimethylation of
lysine 4 of histone H3 is measured as an indication of the methylation
status of the miRNA encoding gene.

[0092]In a related embodiment, occupancy of the miRNA gene by methyl
binding domain containing proteins may be utilised as an indication of
the methylation status of the at least one gene encoding/expression level
of the miRNA. As is shown in the experimental section below,
demethylation of 124a miRNA encoding genes and a consequential
up-regulation in expression is mirrored by an absence of methyl binding
domain containing proteins. In cancer cells, the genes encoding 124a
miRNA are methylated leading to lower levels of 124a miRNA and this is
linked to the presence of methyl binding domain containing proteins in
the CpG island of the 124a miRNA gene. Preferred methyl binding domain
containing proteins include MeCP2 and/or MBD2.

[0093]For these methods in which the CpG island is monitored, preferably
chromatin immunoprecipitation is utilised in order to determine the
respective indication of the methylation status of the at least one gene
encoding the miRNA. Chromatin immunoprecipitation is a well known
technique in the art (see for example Ballestar, E et al. Methyl-CpG
binding proteins identify novel sites of epigenetic inactivation in human
cancer. EMBO J. 22, 6335-6345 (2003)) which relies upon cross-linking of
the binding protein to the DNA, followed by isolation, shearing of the
DNA, antibody detection and isolation by precipitation. The isolated DNA
is then released from the binding protein by reversing the cross-linking
and is amplified by PCR to determine where the binding protein was bound
(Metivier, R. et al., Estrogen receptor-alpha directs ordered, cyclical,
and combinatorial recruitment of cofactors on a natural target promoter,
Cell 2003, 115(6) P751-63). However, any other suitable technique may be
employed as appropriate.

[0094]In one specific embodiment, primers comprising, consisting
essentially of or consisting of the following nucleotide sequences are
used in the chromatin immunoprecipitation methods of the invention:

[0095]Functional derivatives of these primers are also encompassed within
the scope of the present invention. The primers may contain minor
variations, such as single nucleotide substitutions, small additions and
deletions, provided that their ability to act as primers in the
appropriate chromatin immunoprecipitation methods is not compromised.
Thus, variant primers must retain the ability to amplify the appropriate
PCR product. Thus, the primers may be between 10 and 50 nucleotides in
length, preferably between 12 and 40 nucleotides and most preferably
between 15 and 30 nucleotides. The discussion provided in respect of
variants of MSP primers applies mutatis mutandis here.

Methods of Treatment, Compositions Etc.

[0096]In a further aspect the invention provides a method of treating a
condition characterised by methylation of at least one gene encoding an
miRNA comprising administering to a patient in need of treatment a
demethylating agent in an amount effective to restore miRNA
function/expression (by removing methylation of the one or more miRNA
encoding genes). The method preferably comprises administration of a DNA
demethylating agent and/or a DNA methyltransferase inhibitor and/or a
HDAC inhibitor. Preferably, the subject has been selected for treatment
on the basis of determining the methylation status of an miRNA encoding
gene, in particular at least one 124a miRNA gene, according to the
methods of the invention. In an alternative or additional embodiment, the
subject is selected for treatment on the basis of measuring the
expression levels of 124a miRNA according to any of the methods of the
invention.

[0097]Thus, for the patient population where an miRNA encoding gene and
preferably at least one gene encoding 124a miRNA is methylated, which
leads to decreased gene expression, this type of treatment is
recommended. Preferably, treatment involves use of a DNA demethylating
agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor.
This method is referred to hereinafter as the "method of treatment"
aspect of the invention.

[0098]In a related aspect, the invention also provides for the use of a
demethylating agent such as a DNA demethylating agent and/or a DNA
methyltransferase inhibitor and/or a HDAC inhibitor in the manufacture of
a medicament for use in treating a condition characterised by methylation
of one or more genes encoding an miRNA in a subject. This aspect may also
be considered as a DNA methylation agent and/or a DNA methyltransferase
inhibitor and/or a HDAC inhibitor for use in treating a condition
characterised by methylation of one or more genes encoding an miRNA in a
subject. Preferably, the subject has been selected for treatment on the
basis of determining the methylation status of a gene encoding an miRNA,
in particular 124a miRNA, according to any of the (specific) methods of
the invention. Additionally or alternatively, the subject may be selected
for treatment on the basis of measuring the expression levels of an
miRNA, in particular 124a miRNA, according to any of the methods of the
invention.

[0099]Thus, the patient population may be selected for treatment on the
basis of their methylation status with respect to the relevant gene
encoding an miRNA, in particular 124a miRNA which leads to down
regulation of gene expression of the corresponding miRNA. This leads to a
much more focussed and personalised form of medicine and thus leads to
improved success rates since patients will be treated with drugs which
are most likely to be effective. In the experimental section below, it is
shown for the first time that the methylation status of an miRNA gene is
disease associated and may, therefore, be useful for indicating whether
certain treatments such as use of DNA methyltransferase inhibitors and/or
a HDAC inhibitors is likely to prove successful.

[0100]In a related aspect the invention also provides a method of treating
a condition characterised by methylation of one or more genes encoding an
miRNA comprising administering to a patient in need of treatment one or
more miRNAs, wherein the administered miRNAs complement miRNA function
suppressed by methylation in the patient. This may be considered as one
or more miRNAs for use in treating a condition characterised by
methylation of one or more genes encoding an miRNA, wherein the
administered miRNAs complement miRNA function suppressed by methylation
in the patient or as use of one or more miRNAs in the manufacture of a
medicament for treating a condition characterised by methylation of one
or more genes encoding an miRNA, wherein the administered miRNAs
complement miRNA function suppressed by methylation in the patient.

[0101]The invention also provides a method for treating a condition
characterised by methylation of one or more genes encoding an miRNA in a
subject, said subject having a reduced level or activity of the one or
more miRNAs, in particular 124a miRNA, comprising reconstitution of miRNA
expression in the subject. This may be achieved by alleviating
methylation of the relevant miRNA encoding genes or supplying suitable
miRNAs to restore functionality for example, as described herein.

[0102]For the avoidance of doubt, when reference is made to methods of
treatment, reference is intended to also be made to the uses described
herein.

[0103]Thus, methylation of miRNA encoding genes, in particular the 124a
miRNA genes, has been shown for the first time herein to be relevant to
cancer. Methylation is linked to down regulation of miRNA expression.
Accordingly, it is predicted that methods for increasing miRNA expression
will result in improved recovery from cancer in cases where the relevant
miRNA gene is methylated. As shown in the experimental section below,
reconstitution of functional miRNA produces tumour suppressor like
features in transfected cells.

[0104]In all aspects of the invention the most preferred miRNA is 124a
miRNA. Thus, the miRNA comprises 124a miRNA having the sequence
ACCGUAAGUGGCGCACGGAAUU (SEQ ID NO: 1), or a functional derivative
thereof, as defined and described in greater detail herein.

[0105]In a particularly preferred embodiment of the invention, the disease
which is treated is cancer. Preferably, the disease or cancer is not
blastoma, in particular neuroblastoma, or sarcoma. In a particularly
preferred embodiment of the invention, the cancer is selected from colon
cancer, breast cancer, lung cancer, leukaemia and lymphoma. In all of
these disease conditions, methylation of the gene encoding 124a miRNA and
consequential reduced expression of 124a miRNA has been shown. A most
preferred disease condition in which methylation of miRNA genes is
relevant and in which patients may be selected and treated in accordance
with the methods of the invention is colon cancer.

[0106]In methods involving reconstitution of functional miRNA activity, a
recombinant construct capable of expressing an miRNA comprising the
nucleotide sequence ACCGUAAGUGGCGCACGGAAUU (SEQ ID NO: 1) or a functional
derivative thereof with the ability to down-regulate CDK6 expression is
provided. Suitable recombinant constructs may be based upon suitable
constructs known in the art and commercially available. Thus, the
invention provides a recombinant construct capable of directing
transcription of a nucleotide sequence to produce the 124a miRNA
sequence. Variants include all those which retain functionality in terms
of their ability to inhibit translation of the oncogene CDK6. Thus all
functional derivatives of the 124a miRNA sequence which retain the
ability to down-regulate expression of CDK6 at the protein level are
included within the scope of the invention. Functional derivatives
include those having nucleotide additions, substitutions and deletions
and may include non-natural bases as appropriate.

[0107]Recombinant constructs according to the invention may include the
full gene sequence encoding the pri-miRNA and/or pre-miRNA from any
suitable source, although the nucleotide sequence is preferably that from
humans. Preferred is inclusion of the genes
MIRN124A1/MIRN124A2/MIRN124A3. The nucleotide sequence contained within
the recombinant construct may also allow for minor variations provided
that the miRNA finally expressed by the construct retains functionality
in terms of its ability to inhibit CDK6 production. The nucleic acid
molecule which encodes the 124a miRNA may be operably linked to a
suitable regulatory sequence in order to direct specific expression in a
host cell. The nature of the regulatory sequences employed will be
determined at least in part by the nature of the host cell used to
produce the miRNA. The term "regulatory sequence" is to be taken in a
broad context and refers to a regulatory nucleic acid capable of
effecting expression of the nucleic acid molecule to which it is operably
linked. Encompassed by the aforementioned term are promoters and nucleic
acids or synthetic fusion molecules or derivatives thereof which activate
or enhance expression of a nucleic acid, so-called "activators" or
"enhancers". The term "operably linked" as used herein refers to a
functional linkage between the "promoter" sequence and the nucleic acid
molecule of interest, such that the "promoter" sequence is able to
initiate transcription of the nucleic acid molecule to produce the 124a
miRNA.

[0108]A preferred regulatory sequence is a promoter, which may be a
constitutive or an inducible promoter. In a most preferred embodiment,
expression is driven by at least one, preferably one, RNA polymerase
promoter. Preferred promoters are inducible promoters to allow tight
control of expression of the miRNA. Promoters inducible through use of an
appropriate chemical, such as IPTG are preferred. Alternatively, the
transgene encoding the miRNA is placed under the control of a strong
constitutive promoter Preferably, any promoter which is used will direct
strong expression of the miRNA. The nature of the promoter utilised may,
in part, be determined by the specific host cell utilised to produce the
RNA. In one embodiment, the regulatory sequence comprises a bacteriophage
promoter, such as a T7, T3, SV40 or SP6 promoter, most preferably a T7
promoter. In yet other embodiments of the present invention, other
promoters useful for the expression of RNA are used and include, but are
not limited to, promoters from an RNA Pol I, an RNA Pol II or an RNA Pol
III polymerase. Other promoters derived from yeast or viral genes may
also be utilised as appropriate.

[0109]In an alternative embodiment, the regulatory sequence comprises a
promoter selected from the well known tac, trc and lac promoters.
Inducible promoters suitable for use with bacterial hosts include
β-lactamase promoter, E. coli λ phage PL and PR promoters, and
E. coli galactose promoter, arabinose promoter and alkaline phosphatase
promoter. Therefore, the present invention also encompasses a method for
generating 124a miRNA. This method comprises the steps of introducing
(e.g. by transformation, transfection or injection) a recombinant
construct of the invention into a host cell of the invention under
conditions that allow transcription from said recombinant (DNA) construct
to produce the miRNA.

[0110]Optionally, one or more transcription termination sequences or
"terminators" may also be incorporated in the recombinant construct of
the invention. The term "transcription termination sequence" encompasses
a control sequence at the end of a transcriptional unit, which signals 3'
processing and poly-adenylation of a primary transcript and termination
of transcription. As mentioned above, typically miRNAs are produced via
pri- and pre-miRNA transcripts which are polyadenylated. The
transcription termination sequence is useful to prevent read through
transcription such that the miRNA is accurately produced in or by the
host cell. In one embodiment, the terminator comprises a T7, T3, SV40 or
SP6 terminator, preferably a T7 terminator. Other terminators derived
from yeast or viral genes may also be utilised as appropriate.

[0111]Additional regulatory elements, such as transcriptional enhancers,
may be incorporated in the expression construct. The recombinant
constructs of the invention may further include an origin of replication
which is required for maintenance and/or replication in a specific cell
type. One example is when an expression construct is required to be
maintained in a bacterial cell as an episomal genetic element (e.g.
plasmid or cosmid molecule) in a cell. Preferred origins of replication
include, but are not limited to, f1-ori and colE1 ori. The recombinant
construct may optionally comprise a selectable marker gene. As used
herein, the term "selectable marker gene" includes any gene, which
confers a phenotype on a cell in which it is expressed to facilitate the
identification and/or selection of cells, which are transfected or
transformed, with a recombinant construct of the invention. Examples of
suitable selectable markers include resistance genes against ampicillin
(Ampr), tetracycline (Tcr), kanamycin (Kanr), phosphinothricin, and
chloramphenicol (CAT) gene. Other suitable marker genes provide a
metabolic trait, for example manA. Visual marker genes may also be used
and include for example beta-glucuronidase (GUS), luciferase and Green
Fluorescent Protein (GFP).

[0112]In addition, the invention also relates to a host cell comprising a
recombinant construct of the invention. The host cell expressing the
miRNA molecule may be any suitable host cell, including both prokaryotic
and eukaryotic cells. According to one embodiment, any bacterium or
cyanobacterium that is capable of expressing the miRNA molecule from the
recombinant constructs of the invention can be used. The bacterium may be
chosen from the group comprising Gram-negative and Gram-positive
bacteria, such as, but not limited to, Escherichia spp. (e.g. E. coli),
Pseudomonas spp., Enterobacter spp., Bacillus spp. (e.g. B.
thuringiensis), Rhizobium spp., Lactobacilllus spp., Lactococcus spp.,
etc. Preferred bacteria include those from the genus Escherichia, in
particular E. coli and all suitable strains thereof. According to another
embodiment, the host cell comprises a unicellular eukaryote or an
organism from the Kingdom Protista. Unicellular organism for use in the
methods of the invention include, by way of example and not limitation,
algae and yeast. Yeast cells represent a preferred host cell, in
particular well characterised unicellular yeast such as those from the
genus Saccharomyces e.g. S. cerevisiae or Schizosaccharomyces pombe.
Transformation and stable expression can be achieved in yeast cells by
routine methods (for example using YACS, see Sambrook and
Russell--Molecular Cloning: A laboratory manual--third edition and the
references provided therein in Protocol 10). A yeast recombinant
construct can typically include one or more of the following: a promoter
sequence, fusion partner sequence, leader sequence, transcription
termination sequence, a selectable marker. These elements can be combined
into an expression cassette, which may be maintained in a replicon, such
as an extrachromosomal element (e.g., plasmids) capable of stable
maintenance in a host, such as yeast or bacteria. The replicon may have
two replication systems, thus allowing it to be maintained, for example,
in yeast for expression and in a prokaryotic host for cloning and
amplification. Examples of such yeast-bacteria shuttle vectors include
YEp24 (Botstein et al., 1979), pCI/1 (Brake et al., 1984), and YRp17
(Stinchcomb et al., 1982). In addition, a replicon may be either a high
or low copy number plasmid. A high copy number plasmid will generally
have a copy number ranging from about 5 to about 200, and typically about
10 to about 150. A host containing a high copy number plasmid will
preferably have at least about 10, and more preferably at least about 20.
Useful yeast promoter sequences can be derived from genes encoding
enzymes in the metabolic pathway. Examples of such genes include alcohol
dehydrogenase (ADH) (EP 0 284044), enolase, glucokinase,
glucose-6-phosphate isomerase, glyceraldehyde-3-phosphatedehydrogenase
(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate
mutase, and pyruvate kinase (PyK). The yeast PHO5 gene, encoding acid
phosphatase, also provides useful promoter sequences (Myanohara et al.,
1983). In addition, synthetic promoters that do not occur in nature also
function as yeast promoters. Examples of such hybrid promoters include
the ADH regulatory sequence linked to the GAP transcription activation
region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Examples of
transcription terminator sequences and other yeast-recognized termination
sequences, such as those coding for glycolytic enzymes, are known to
those of skill in the art.

[0113]Alternatively, the expression constructs can be integrated into the
yeast genome with an integrating vector. Integrating vectors typically
contain at least one sequence homologous to a yeast chromosome that
allows the vector to integrate, and preferably contain two homologous
sequences flanking the expression construct. Integrations appear to
result from recombinations between homologous DNA in the vector and the
yeast chromosome (Orr-Weaver et al., 1983). An integrating vector may be
directed to a specific locus in yeast by selecting the appropriate
homologous sequence for inclusion in the vector (Orr-Weaver et al.,
1983).

[0114]In one embodiment, the host cells are inactivated following
expression of the miRNA. These host cells may then be utilised in the
methods of treatment of the invention together with suitable excipients
or carriers or diluents Suitable inactivation techniques include heat
treatment or chemical treatment, for example through use of phenol or
formaldehyde.

[0115]Other suitable host cells include those derived from animals such as
CHO cells etc., which are routinely used to express products for
pharmaceutical use. Examples of mammalian cells which may be used to
produce miRNA include the tetracycline resistant cells commercially
available from Invitrogen. The miRNA once expressed by any suitable host
cell may be isolated prior to use in the methods of treatment of the
invention. This may be achieved by any suitable means such as by routine
RNA extraction followed by gel purification or through use of sequence
specific hybridization techniques for example, as would be readily
apparent to one of skill in the art.

[0116]Such recombinant constructs and host cells may be used to deliver
wild type copies of at least one gene encoding an miRNA, and preferably
the at least one genes encoding 124a miRNA, into the subject. However,
any functional derivative of the miRNA may be utilised provided the
desired therapeutic effect is achieved.

[0117]Any suitable vector for delivery of functional copies of at least
one gene encoding an miRNA, and preferably the at least one genes
encoding 124a miRNA, may be utilised according to the method of the
invention where expression of the miRNA occurs in vivo. One principal
requirement is that tissue specificity of delivery and expression is
achieved. The two major sources of vectors which may be utilised comprise
viral vectors and non-viral vectors.

[0118]Within the group of viral vectors, preferred types include
adenoviruses, retroviruses, in particular Moloney murine leukaemia virus
(Mo-MLV), adeno-related viruses and herpes simplex virus type I.
Typically, the gene of interest, in this case encoding at least one
miRNA, and preferably at least 124a miRNA, will be included in the viral
genome, preferably in the "non-essential" region of the viral genome. In
addition, it is important to remove virally encoded proto-oncogenes from
the viral vector genome. The virus may be made replication incompetent to
prevent unwanted replication once the virus has been targeted.

[0119]In terms of targeting the viral vector to the desired site, which
depends upon the specific disease to be treated, a number of
possibilities exist. For example, the env gene (which encodes the viral
vector's envelope) may be engineered or replaced with the env gene from a
different virus to alter the range of cells the viral vector will
"infect". Furthermore, alteration of the viral tropism may be achieved by
using suitable antibodies raised against antigenic determinants on the
cell surface of the desired target cells. The antibodies, which include
all derivatives thereof, such as scFV, nanobodies, VH domains, Fab
fragments etc., may be genetically incorporated into the viral vectors to
provide targeted gene delivery of the at least one gene encoding an
miRNA, and preferably the at least one genes encoding 124a miRNA. Most
preferred is use of scFV (Hedley et al., Gene Therapy (2006) 13, 88-94).
The viral vectors may have many genes removed, such as packaging genes,
in order to reduce immunogenicity and/or infectivity. These functions may
thus be supplied by a helper virus.

[0120]Due to their high efficiency of integration, low pathogenicity and
high efficacy, adenoviruses are a preferred vector according to the
methods of the invention.

[0121]Alternatives to viral vectors include direct gene delivery, use of
other delivery agents and use of molecular conjugates. Tissue specific
promoters may be employed as appropriate. Direct gene delivery may be
achieved for example by microinjection of a suitable vector, such as a
recombinant construct of the invention, directly into the tissue of
interest. Alternatives include use of ballistic transformation, for
example using vector coated onto suitable particles (e.g. gold
particles). Additional delivery agents include liposomes and derivatives
thereof. As discussed above, targeting proteins such as antibodies and
derivatives thereof may be utilised in order to ensure delivery to the
cells of interest. Molecular conjugates may include suitable proteins
conjugated to the DNA of interest using a suitable DNA binding agent.

[0122]It is important that the nucleic acid molecules or gene copies
provided in the recombinant constructs, host cells and other vectors of
the invention are unmethylated in order to ensure maximal expression of
the miRNA to provide a therapeutic effect. One method of preventing
methylation of the miRNA encoding nucleic acid is to also incorporate a
sequence encoding an inhibitor of DNA methyltransferase activity into the
recombinant construct. Thus, suitable antisense or siRNA molecules may
also be encoded by the recombinant construct for example, which when
expressed down regulate any DNA methyltransferase activity present by RNA
interference. Down regulation may occur through DNMT mRNA destruction or
prevention of DNMT translation dependent upon the RISC complex involved.
Alternatively expression may occur in suitable DKO cells, as described in
more detail in the experimental section below, which include a double
knockout of both DNMT1 and DNMT3b and which are therefore hypomethylated.

[0123]In a related aspect, the invention provides for the use of a vector
or host cell carrying at least one gene encoding an miRNA, and preferably
the at least one gene encoding 124a miRNA, in the manufacture of a
medicament for treating a condition in which an miRNA is methylated, and
preferably cancer, in a subject. This may also be expressed as a vector
or host cell carrying at least one gene encoding an miRNA for use in
treating a condition, such as cancer, in which an miRNA is methylated.
Preferably, the subject has been selected for treatment according to the
methods of the invention. Methylation of the at least one gene encoding
an miRNA, and preferably the at least one genes encoding 124a miRNA
and/or a decreased level of miRNA expression selects the subject for
treatment. Thus, methylation may, in one embodiment, be determined at the
level of gene expression.

[0124]In a preferred embodiment, the reduced level or activity of the
miRNA, in particular 124a miRNA, is detected by determining the
methylation status of the at least one gene encoding an miRNA, and
preferably the at least one genes encoding 124a miRNA. This may be done
according to any of the methods of the invention described. Thus, a
particular subgroup of subjects suffering from, or predicted to have a
likelihood of developing, a particular disease such as cancer is selected
for treatment according to whether the at least one gene encoding an
miRNA, and preferably the at least one genes encoding 124a miRNA, is
methylated or not (which may be determined at the level of gene
expression if desired).

[0125]As mentioned above, in a particularly preferred embodiment of the
invention, the disease which is treated is cancer. Preferably, the
disease or cancer is not blastoma, in particular neuroblastoma, or
sarcoma. In a particularly preferred embodiment of the invention, the
cancer is selected from colon cancer, breast cancer, lung cancer,
leukaemia and lymphoma. A most preferred disease condition in which
methylation of miRNA genes is relevant and in which patients may be
selected and treated in accordance with the methods of the invention is
colon cancer.

[0126]For all of the relevant aspects (such as methods of treatment) of
the invention, as appropriate the DNA demethylating agent may be any
agent capable of up regulating transcription of at least one miRNA, and
preferably at least 124a miRNA. A preferred DNA demethylating agent
comprises, consists essentially of or consists of a DNA methyltransferase
inhibitor. For all of the relevant methods of the invention, the DNA
methyltransferase inhibitor may be any suitable inhibitor of DNA
methyltransferase which is suitable for treating cancer in the presence
of methylation of the at least one gene encoding an miRNA, and preferably
the at least one genes encoding 124a miRNA. As is shown in the
experimental section below, methylation of the genes encoding 124a miRNA
is linked to the incidence of cancer and so preventing this methylation
is predicted to help to treat cancer.

[0127]The DNA methyltransferase inhibitor may, in one embodiment, be one
which reduces expression of DNMT genes, such as suitable antisense
molecules, or siRNA molecules which mediate RNAi for example. The design
of a suitable siRNA molecule is within the capability of the skilled
person and suitable molecules can be made to order by commercial entities
(see for example, www.ambion.com). Preferably, the DNA methyltransferase
gene is (human) DNMT1.

[0128]Alternatively, the agent may be a direct inhibitor of DNMTs.
Examples include modified nucleotides such as phosphorothioate modified
oligonucleotides (as set out in FIG. 6 of Villar-Garea, A. And Esteller,
M. DNA demethylating agents and chromatin-remodelling drugs: which, how
and why? Current Drug Metabolism, 2003, 4, 11-31, which reference is
hereby incorporated in its entirety) and nucleosides and nucleotides such
as cytidine analogues. Suitable examples of cytidine analogues include
5-azacytidine, 5-aza-2'-deoxycytidine, 5-fluoro-2'-deoxycytidine,
pseudoisocytidine, 5,6-dihydro-5-azacytidine,
1-β-D-arabinofuranosyl-5-azacytosine (known as fazabarine) (see FIG.
4 of Villar-Garea, A. And Esteller, M. DNA demethylating agents and
chromatin-remodelling drugs: which, how and why? Current Drug Metabolism,
2003, 4, 11-31, which reference is hereby incorporated in its entirety).

[0129]In another embodiment, the DNA methyltransferase inhibitor comprises
Decitabine. Full details of this drug can be found at www.supergen.com
for example.

[0131]Further agents which may alter DNA methylation and which may,
therefore, be useful in the present compositions include
organohalogenated compounds such as chloroform etc, procianamide,
intercalating agents such as mitomycin C, 4-aminobiphenyl etc, inorganic
salts of arsenic and selenium and antibiotics such as kanamycin,
hygromycin and cefotaxim (Villar-Garea, A. And Esteller, M. DNA
demethylating agents and chromatin-remodelling drugs: which, how and why?
Current Drug Metabolism, 2003, 4, 11-31).

[0132]However, any suitable DNA methyltransferase inhibitor which is
capable of increasing the expression of at least one gene encoding an
miRNA, and preferably the at least one genes encoding 124a miRNA, and
thus can contribute to the treatment of cancer, is included within the
scope of the invention.

[0134]For all of the relevant methods of the invention, the histone
deacetylase (HDAC) inhibitor may be any suitable inhibitor of HDAC
activity which is suitable for treating cancer in the presence of
methylation of at least one gene encoding an miRNA, and preferably the at
least one genes encoding 124a miRNA.

[0136]The methods of treatment and medical uses aspects of the invention
may incorporate any and all of the preferred aspects described in respect
of the other methods of the invention as described above. Preferably, the
diagnostic methods and/or the pharmacogenetic methods and/or the
treatment regimen methods of the invention are carried out as a prelude
to, or as an integral part of the methods of treating cancer according to
the gene therapy aspects of the invention. The gene therapy methods may
be synergistically combined with those of the methods of treatment
according to the invention.

[0137]Thus, for example, the description of suitable methods for
determining methylation levels of at least one gene encoding an miRNA,
and preferably the at least one genes encoding 124a miRNA, suitable test
samples, preferred subjects and specific types of cancer which may be
treated all apply mutatis mutandis to these aspects of the invention and
are not repeated here simply for reasons of conciseness.

[0138]Due to the effect of 124a miRNA on the oncogene CDK6's activity, the
invention provides a method of treating cancer in a patient comprising
reducing CDK6 levels and/or activity. Preferred cancer types are
described above and may be selected from colon cancer, breast cancer,
lung cancer, leukaemia and lymphoma, most preferably colon cancer. In a
preferred embodiment of this method of the invention, the levels and/or
activity of CDK6 are reduced by removing methylation of one or more genes
encoding specified miRNA molecules which act to inhibit expression of
CDK6 and/or by administering to the patient one or more miRNAs in an
effective amount to the patient, wherein the administered miRNAs act to
reduce the levels and/or activity of CDK6 in the patient. In a most
preferred embodiment, the one or more genes encoding miRNA molecules
whose methylation is removed comprises one or more genes encoding 124a
miRNA. Similarly, preferably the one or more miRNA molecules which are
administered comprise 124a miRNA (SEQ ID NO: 1 ACCGUMGUGGCGCACGGAAUU) or
a functional derivative thereof with the ability to down-regulate CDK6
(protein) expression. Functional derivatives include all those retaining
the ability to repress CDK6 translation, as discussed in greater detail
above. The methods may be considered as medical use embodiments, in
accordance with the other methods of the invention.

[0139]In a related aspect of the invention, there is provided a
pharmaceutical composition comprising an miRNA together with a suitable
carrier/excipient/diluent. Prior to the present invention,
down-regulation of miRNA expression due to methylation had not been
proven with a disease association. Accordingly, the inclusion of an miRNA
in a pharmaceutical composition represents a preferred aspect of the
present invention in order to allow recovery of miRNA expression levels
in a subject in need of treatment. Preferably, the pharmaceutical
composition includes 124a miRNA (SEQ ID NO: 1 ACCGUMGUGGCGCACGGAAUU) or a
functional derivative thereof with the ability to down-regulate CDK6
expression, preferably protein expression (as defined above). In a
further embodiment, the composition comprises a recombinant construct of
the invention and/or a host cell of the invention and/or a vector of the
invention.

[0140]It should be noted that, for the purposes of the present invention,
the designation of a particular miRNA containing or producing composition
is considered to encompass all pharmaceutically acceptable forms of the
active compound which are useful in methods of treating conditions where
specific genes encoding miRNAs are methylated, such as cancer. Thus,
stereoisomers, enantiomers, salts, esters etc are all encompassed within
the scope of the invention as appropriate.

[0141]Thus, in a pharmaceutical composition of the invention, preferred
compositions include pharmaceutically acceptable carriers including, for
example, non-toxic salts, sterile water or the like. A suitable buffer
may also be present allowing the compositions to be lyophilized and
stored in sterile conditions prior to reconstitution by the addition of
sterile water for subsequent administration. The carrier may also contain
other pharmaceutically acceptable excipients for modifying other
conditions such as pH, osmolarity, viscosity, sterility, lipophilicity,
somobility or the like. Pharmaceutical compositions which permit
sustained or delayed release following administration may also be used.
The composition may optionally incorporate suitable RNase inhibitors to
prevent degradation of the miRNAs.

[0142]Suitable pharmaceutical compositions for use in the treatment
methods or medical uses of the invention may be used together with other
standard chemotherapeutic treatments which target tumour cells directly.
Non limiting examples include paclitaxel, cyclaphosphomide and
5-tumor-uracil (5-FU) and pharmaceutically acceptable derivatives thereof
including salts, etc.

[0143]The miRNA containing pharmaceutical composition may, for example, be
encapsulated and/or combined with suitable carriers in solid dosage forms
for oral administration which would be well known to those of skill in
the art or alternatively with suitable carriers for administration in an
aerosol spray. Examples of oral dosage forms include tablets, capsules
and liquids.

[0144]Alternatively, the therapeutic agent may be administered
parenterally. Specific examples include intradermal injection,
subcutaneous injection (which may advantageously give slower absorption
of the therapeutic agent), intramuscular injection (which can provide
more rapid absorption), intravenous delivery (meaning the drug does not
need to be absorbed into the blood stream from elsewhere), sublingual
delivery (for example by dissolving of a tablet under the tongue or by a
sublingual spray), rectal delivery, vaginal delivery, topical delivery,
transdermal delivery and inhalation.

[0145]Furthermore, as would be appreciated by the skilled practitioner,
the specific dosage regime may be calculated according to the body
surface area of the patient or the volume of body space to be occupied,
dependent on the particular route of administration to be used. The
amount of the composition actually administered will, however, be
determined by a medical practitioner based on the circumstances
pertaining to the disorder to be treated, such as the severity of the
symptoms, the age, weight and response of the individual.

[0146]Thus, in a related aspect there is also provided miRNA molecules for
use as a medicament. Thus, specific miRNA molecules may be of therapeutic
use to treat specific conditions where methylation of an miRNA encoding
gene leads to lower levels of expression of the miRNA. In a more specific
embodiment, there is provided 124a miRNA (SEQ ID NO: 1
ACCGUMGUGGCGCACGGAAUU) or a functional derivative thereof with the
ability to down-regulate CDK6 (protein) expression, for use as a
medicament. 124a miRNA is a most preferred miRNA for incorporation into a
medicament due to the link shown herein between methylation of the
different genes encoding this miRNA and various types of cancer.
Similarly, the invention therefore also provides for use of an miRNA in
the manufacture of a medicament for the treatment of cancer. Preferably,
and as discussed in more detail above, the cancer is selected from colon
cancer, breast cancer, lung cancer, leukaemia and lymphoma. The cancer is
most preferably colon cancer. Preferably, the miRNA comprises 124a miRNA
(SEQ ID NO: 1 ACCGUAAGUGGCGCACGGAAUU) or a functional derivative thereof
with the ability to down-regulate CDK6 (protein) expression.

Kits of the Invention

[0147]The invention also provides kits which may be used in order to carry
out the methods of the invention. The kits may incorporate any of the
preferred features mentioned in connection with the various methods and
uses of the invention above, which discussion applies here mutatis
mutandis.

[0148]Thus, a kit is provided for: [0149](a) diagnosing a disease in
which an miRNA is expressed at a lower level; and/or [0150](b) predicting
the likelihood of successful treatment of a disease in which an miRNA is
expressed at a lower level and/or the likelihood of resistance to
treatment of a disease in which an miRNA is expressed at a lower level
with a DNA demethylating agent and/or a DNA methyltransferase inhibitor
and/or a HDAC. inhibitor, and/or [0151](c) selecting a suitable treatment
regimen for a disease in which an miRNA is expressed at a lower
levelcomprising carrier means containing therein a set of primers for use
in detecting the methylation status of at least one gene encoding the
miRNA which is expressed at a lower level in the disease. As
aforementioned, the most preferred miRNA is 124a miRNA.

[0152]This kit is preferably a kit for use in MSP and even more preferably
a real-time or end point detection version of MSP.

[0153]Thus, the kit includes suitable primers for determining whether the
at least one gene encoding an miRNA, and preferably the at least one
genes encoding 124a miRNA is methylated. These primers may comprise any
of the primers discussed in detail in respect of the various methods of
the invention which may be employed in order to determine the methylation
status of the at least one gene encoding an miRNA, and preferably the at
least one genes encoding 124a miRNA. As mentioned above, functional
derivatives of the specific primers may also be incorporated into the
kits of the invention.

[0154]In one embodiment, the kit of the invention further comprises a
reagent which modifies unmethylated cytosine in a detectable fashion
whilst leaving methylated cytosine residues in tact. Such a reagent is
useful for distinguishing methylated from unmethylated cytosine residues.
In a preferred embodiment, the reagent comprises bisulphite, disulphite
or hydrogen sulphite and preferably sodium bisulphite. This reagent is
capable of converting unmethylated cytosine residues to uracil whereas
methylated cytosines remain unconverted. This difference in residue may
be utilised to distinguish between methylated and unmethylated nucleic
acid in a downstream process, such as PCR using primers which distinguish
between cytosine and uracil (cytosine pairs with guanine, whereas uracil
pairs with adenine).

[0155]In a real-time and/or end point detection embodiments, the kit may
further comprise probes for real-time detection of amplification
products. These probes may simply be used to monitor progress of the
amplification reaction in real-time and/or they may also have a role in
determining the methylation status of the at least one gene encoding an
miRNA, and preferably the at least one genes encoding 124a miRNA,
themselves. Thus, the probes may be designed in much the same fashion as
the primers to take advantage of sequence differences following treatment
with a suitable reagent such as sodium bisulphite dependent upon the
methylation status of the appropriate cytosine residues (found in CpG
dinucleotides).

[0157]All of these technologies are well characterised in the art and the
design of suitable probes is routine for one of skill in the art.
Notably, however, with the AMPLIFLUOR and SCORPION embodiments, the
probes are an integral part of the primers which are utilised. The probes
are typically fluorescently labelled, although other label types may be
utilised as appropriate.

[0158]In one embodiment, the primers in the kit comprise, consist
essentially of, or consist of primers which are capable of amplifying
methylated and/or unmethylated DNA following bisulphite treatment which
DNA comprises, consists essentially of, or consists of the nucleotide
sequence of the gene or genes encoding 124a miRNA. These genes are found
at the genomic loci 8p23.1/8q12.3/20q13.33 and are known as MIRN124A1,
MIRN124A2 and MIRN124A3 respectively.

[0159]In a specific embodiment, the primers in the kit comprise, consist
essentially of, or consist of primers comprising, consisting essentially
of, or consisting of the following nucleotide sequences for the purposes
of amplifying methylated DNA (following bisulphite treatment):

[0160]In a specific embodiment, the primers in the kit comprise, consist
essentially of, or consist of primers comprising, consisting essentially
of, or consisting of the following nucleotide sequences for the purposes
of amplifying unmethylated DNA (following bisulphite treatment):

[0161]Functional derivatives of these sequences (as defined herein) are
explicitly included within the scope of the invention.

[0162]In a further embodiment, in which bisulphite sequencing is utilised
in order to determine the methylation status of the at least one gene
encoding an miRNA, and preferably the at least one genes encoding 124a
miRNA, the kit comprises primers for use in sequencing through the
important CpG islands in the at least one gene encoding an miRNA, and
preferably the at least one genes encoding 124a miRNA. Thus, primers may
be designed in both the sense and antisense orientation to direct
sequencing across the promoter region of the gene.

[0163]In one embodiment, the primers in the kit comprise, consist
essentially of, or consist of primers which are capable of sequencing of
DNA following bisulfite treatment which DNA comprises, consists
essentially of, or consists of the nucleotide sequence of the gene or
genes encoding 124a miRNA. These genes are found at the genomic loci
8p23.1/8q12.3/20q13.33 and are known as MIRN124A1, MIRN124A2 and
MIRN124A3 respectively.

[0164]In one embodiment, bisulphite sequencing may be carried out by using
sequencing primers which comprise, consist essentially of or consist of
the following sequences, and which may be used in isolation or in
combination to sequence both strands:

[0165]Again, functional derivatives of these sequences, which retain
usefulness in bisulphite sequencing (of one or more genes encoding 124a
miRNA) are included within the scope of the invention. Thus, the
discussion above applies mutatis mutandis here.

[0166]As discussed with respect to the methods of the invention, suitable
controls may be utilised in order to act as quality control for the
methods. Accordingly, in one embodiment, the kit of the invention further
comprises, consists essentially of or consists of one or more control
nucleic acid molecules of which the methylation status is known. These
(one or more) control nucleic acid molecules may include both nucleic
acids which are known to be, or treated so as to be, methylated and/or
nucleic acid molecules which are known to be, or treated so as to be,
unmethylated. One example of a suitable internal reference gene, which is
generally unmethylated, but may be treated so as to be methylated, is
β-actin.

[0167]Furthermore, the kit of the invention may further comprise, consist
essentially of or consist of primers for the amplification of the control
nucleic acid. These primers may be the same primers as those utilised to
monitor methylation in the test sample in a preferred embodiment. Thus,
the control nucleic acid may comprise at least one gene encoding an
miRNA, and preferably the at least one genes encoding 124a miRNA, for
example taken from normal tissues (such as normal colon) in which it is
known to be unmethylated. The control nucleic acid may additionally
comprise at least one gene encoding an miRNA, and preferably the at least
one genes encoding 124a miRNA in methylated form, for example as
methylated by a methyltransferase enzyme such as SssI methyltransferase
for example.

[0168]Suitable probes for use in determining the methylation status of the
control nucleic acid molecules may also be incorporated into the kits of
the invention. The probes may comprise any suitable probe type for
real-time or end-point detection of amplification products. Non-limiting
examples include hairpin primers (Amplifluor)/hairpin probes (Molecular
Beacons)/hydrolytic probes (Taqman)/FRET probe pairs
(Lightcycler)/primers incorporating a hairpin probe (Scorpion)/primers
incorporating complementary sequences of DNAzymes that cleave a reporter
substrate included in the reaction mixture (DzyNA®/fluorescent dyes
(SYBR Green etc.)/oligonucleotide blockers/the specific interaction
between two modified nucleotides (Plexor). All of these technologies are
well characterised in the art and the design of suitable probes is
routine for one of skill in the art. Notably, however, with the
AMPLIFLUOR and SCORPION embodiments, the probes are an integral part of
the primers which are utilised. Similarly, kits may include SYBR Green
reagent to allow real time or end point detection of amplification
products.

[0169]The kits of the invention may additionally include suitable buffers
and other reagents for carrying out the claimed methods of the invention.
Thus, the discussion provided in respect of the methods of the invention
as to the requirements for determination of the methylation status of at
least one gene encoding an miRNA, and preferably the at least one genes
encoding 124a miRNA, apply mutatis mutandis here.

[0170]In one embodiment, the kit of the invention further comprises,
consists essentially of, or consists of nucleic acid amplification
buffers.

[0171]The kit may also additionally comprise, consist essentially of or
consist of enzymes to catalyze nucleic acid amplification. Thus, the kit
may also additionally comprise, consist essentially of or consist of a
suitable polymerase for nucleic acid amplification.

[0172]Examples include those from both family A and family B type
polymerises, such as Taq, Pfu, Vent etc.

[0173]In a related aspect of the invention, a kit is provided for:
[0174](a) diagnosing a disease in which an miRNA is expressed at a lower
level; and/or [0175](b) predicting the likelihood of successful treatment
of a disease in which an miRNA is expressed at a lower level and/or the
likelihood of resistance to treatment of a disease in which an miRNA is
expressed at a lower level with a DNA demethylating agent and/or a DNA
methyltransferase inhibitor and/or a HDAC inhibitor, and/or [0176](c)
selecting a suitable treatment regimen for a disease in which an miRNA is
expressed at a lower levelcomprising carrier means containing therein a
set of primers for use in detecting the expression level of an miRNA
which is expressed at a lower level in the disease. As aforementioned,
the most preferred miRNA is 124a miRNA. The most preferred disease is
cancer, as discussed above. Specific preferred cancers are colon cancer,
breast cancer, lung cancer, leukaemia and lymphoma, preferably colon
cancer.

[0177]This kit is preferably a kit for use in RT-PCR and even more
preferably a real-time detection version of RT-PCR.

[0178]Thus, the kit includes suitable primers for determining whether the
miRNA, and preferably 124a miRNA is expressed at a low level in the
sample. These primers may comprise any of the primers discussed in detail
in respect of the various methods of the invention which may be employed
in order to determine the expression levels of the miRNA, and preferably
at least 124a miRNA. As mentioned above, functional derivatives of the
specific primers may also be incorporated into the kits of the invention.

[0179]In a real-time or end point detection embodiment, the kit may
further comprise probes for real-time detection of amplification
products. These probes are used to monitor progress of the amplification
reaction in real-time or at end point.

[0180]The probes may comprise any suitable probe type for real-time or end
point detection of amplification products. Non-limiting examples include
hairpin primers (Amplifluor)/hairpin probes (Molecular
Beacons)/hydrolytic probes (Taqman)/FRET probe pairs
(Lightcycler)/primers incorporating a hairpin probe (Scorpion)/primers
incorporating complementary sequences of DNAzymes that cleave a reporter
substrate included in the reaction mixture (DzyNA®)/fluorescent dyes
(SYBR Green etc.)/oligonucleotide blockers/the specific interaction
between two modified nucleotides (Plexor). All of these technologies are
well characterised in the art and the design of suitable probes is
routine for one of skill in the art. Notably, however, with the
AMPLIFLUOR and SCORPION embodiments, the probes are an integral part of
the primers which are utilised. The probes are typically fluorescently
labelled, although other label types may be utilised as appropriate.

[0181]In one embodiment, the primers in the kit comprise, consist
essentially of, or consist of primers which are capable of amplifying
reverse transcribed RNA, producing cDNA, which RNA comprises, consists
essentially of, or consists of the nucleotide sequence of the pri-miRNA,
pre-miRNA or mature 124a miRNA. The genes encoding the miRNAs, in various
forms from primary transcript through to processed final miRNA molecules
are found at the genomic loci 8p23.1/8q12.3/20q13.33 and are known as
MIRN124A1, MIRN124A2 and MIRN124A3 respectively.

[0182]In a specific embodiment, the primers in the kit comprise, consist
essentially of, or consist of primers comprising, consisting essentially
of, or consisting of the following nucleotide sequences for the purposes
of amplifying cDNA:

[0183]Functional derivatives of these sequences are also envisaged. The
primers may contain minor variations, such as single nucleotide
substitutions, small additions and deletions, provided that their ability
to act as RT-PCR primers is not compromised. Thus, variant primers must
retain the ability to allow determination of expression of 124a miRNA
through RT-PCR techniques. Thus, the primers may be between 10 and 50
nucleotides in length, preferably between 12 and 40 nucleotides and most
preferably between 15 and 30 nucleotides.

[0184]In a preferred embodiment, the RT-PCR is carried out in real time or
at end point and in a quantitative manner. Real time quantitative RT-PCR
has been thoroughly described in the literature (see Gibson et al for an
early example of the technique) and a variety of techniques are possible.
Examples include use of hairpin primers (Amplifluor)/hairpin probes
(Molecular Beacons)/hydrolytic probes (Taqman)/FRET probe pairs
(Lightcycler)/primers incorporating a hairpin probe (Scorpion)/primers
incorporating complementary sequences of DNAzymes that cleave a reporter
substrate included in the reaction mixture (DzyNA®)/fluorescent dyes
(SYBR Green etc.)/oligonucleotide blockers/the specific interaction
between two modified nucleotides (Plexor). systems. All of these systems
are commercially available and well characterised, and may allow
multiplexing (that is, the determination of expression of multiple genes
in a single sample).

[0185]In one specific embodiment, real-time RT-PCR is carried out using
SYBR Green as the detection reagent. This reagent is commercially
available as a PCR master mix from Applied Biosystems. In one specific
embodiment, the kit for real time RT-PCR includes an antisense
oligonucleotide comprising, consisting essentially of or consisting of
the nucleotide sequence GCGAGCACAGAATTAATACGACTC (SEQ ID NO: 37),
optionally together with a sense primer comprising, consisting
essentially of or consisting of the nucleotide sequence TTA AGG CAC GCG
GTG MT GCC A (miR-124a) (SEQ ID NO: 38). Again, functional derivatives
which retain function in the real-time RT-PCR assay are envisaged within
the kits of the invention (as defined above).

[0186]As discussed with respect to the methods of the invention, suitable
controls may be utilised in order to act as quality control for the
methods. Accordingly, in one embodiment, the kit of the invention further
comprises, consists essentially of or consists of one or more control
nucleic acid molecules of which the expression levels are known. One
example of a suitable internal reference gene is β-actin.

[0187]Furthermore, the kits of the invention may further comprise, consist
essentially of or consist of primers for the amplification of the control
nucleic acid. These primers may be the same primers as those utilised to
monitor expression in the test sample in a preferred embodiment. In one
embodiment, primers for determining expression levels of β-actin are
included within the kits of the invention.

[0188]The kits of the invention may additionally include suitable buffers
and other reagents for carrying out the claimed methods of the invention.
Thus, the discussion provided in respect of the methods of the invention
as to the requirements for determination of the expression levels of at
least one miRNA, and preferably at least 124a miRNA, apply mutatis
mutandis here.

[0189]In one embodiment, the kit of the invention further comprises,
consists essentially of, or consists of nucleic acid amplification
buffers.

[0190]The kit may also additionally comprise, consist essentially of or
consist of enzymes to catalyze nucleic acid amplification. Thus, the kit
may also additionally comprise, consist essentially of or consist of a
suitable polymerase for nucleic acid amplification. Examples include
those from both family A and family B type polymerises, such as Taq, Pfu,
Vent etc. The kits may also include suitable reverse transcriptase
enzymes for reverse transcription of mRNA (including pri- and pre-miRNAs)
and also suitable primers for priming reverse transcription.

[0191]The kits of the invention may additionally incorporate the specific
components required for additional analysis by chromatin
immunoprecipitation and/or for determining CDK6 expression and/or for
determining Rb phosphorylation as appropriate. In these embodiments, the
description above in respect of the various methods applies mutatis
mutandis to the kits of the invention which may incorporate the
appropriate reagents.

[0192]The kits of the invention may separately or additionally contain a
pharmaceutical composition of the invention and/or a DNA methylation
agent and/or a DNMT inhibitor or HDAC inhibitor for use depending upon
the determination of the methylation status of at least one gene encoding
an miRNA, and preferably the at least one genes encoding 124a miRNA
and/or the expression levels of at least one miRNA, especially 124a
miRNA. Thus, if the kits of the invention prove a positive result in
terms of methylation and/or resultant down-regulation of expression, the
compositions in the kit may then be employed as a means of treating the
condition, which is preferably cancer as discussed and defined above.

[0193]The various components of the kit may be packaged separately in
separate compartments or may, for example be stored together where
appropriate.

[0194]The kit may also incorporate suitable instructions for use, which
may be printed on a separate sheet or incorporated into the kit packaging
for example.

[0195]The invention will now be described with respect to the following
non-limiting examples.

[0199]FIG. 1c Methylation specific PCR analyses for miR-124a methylation
in primary colorectal tumors. Pairs of normal colon vs colorectal tumors
from three different patients are shown. The presence of a band under the
U or M lanes indicates Unmethylated or Methylated sequences,
respectively. Normal lymphocytes (NL) and In Vitro methylated DNA (IVD)
are shown as unmethylated and methylated control sequences, respectively.

[0202]FIG. 1F Chromatin immunoprecipitation assay for histone modification
marks and methyl-CpG binding domain proteins (MeCP2 and MBD2) in the
miR-124a CpG island. The presence of miR-124a CpG island methylation is
associated with the lack of histone modifications linked to
transcriptional activity (acetylation of histones H3 and H4, and
trimethylation of lysine 4 of histone H3) and with the occupancy by MBDs,
whilst the opposite scenario is observed when DNA demethylation events
are present by genetic disruption of the DNA methylatransferases (DKO
cells) or pharmacological treatment with a DNA demethylating agent (AZA).

[0207]FIG. 2D--miR-124a interact and interfere with the 3' UTR of CDK6
mRNA. 8 luciferase assays of DKO (miR-124a expressor) or HCT-116
(miR-124a non-expressor) cells transfected with firefly luciferase
constructs containing CDK6-WT or CDK-MUT. In the case of HCT-116 cells,
the results of miR-124a cotransfection are also shown. Normalized
luciferase activities are represented.

[0208]FIG. 2E--Western blots for CDK6 in original HCT-116 cells and
HCT-116 cells transfected with miR-124a precursor, demonstrating CDK6
down-regulation. RT-PCR analyses of CDK6 mRNA do not demonstrate any
change.

[0209]FIG. 2F--Western blot for phosphorylated Rb demonstrates that
HCT-116 cells transfected with the miR-124a precursor, HCT-116 cells
treated with the DNA demethylating agent, and DKO cells are
hypophosphorylated at the two Rb sites mediated by CDK6.

[0220]FIG. 5B--Methylation-specific PCR analyses for miR-124a methylation
in human cancer cell lines. The presence of a band under the U or M lanes
indicates Unmethylated or Methylated sequences, respectively. Normal
lymphocytes (NL) and In Vitro methylated DNA (IVD) are shown as negative
and positive controls for unmethylated and methylated sequences,
respectively.

[0223]To explore the putative presence of DNA methylation
associated-silencing of miRNAs in cancer cells, we used a genetic
approach. We compared the miRNA expression profile of the wild-type colon
cancer cell line HCT-116 with the same cell line after genetic disruption
by homologous recombination of DNMT1 and DNMT3b (Double Knock-Out,
DKO)(11), using a miRNA microarray expression profiling method (12). DKO
cells show a drastic reduction of DNMT activity, 5-methylcytosine DNA
content and, most important, a release of gene silencing associated with
CpG island hypomethylation (11,13). Our genetic screening revealed that
eighteen of the three hundred and twenty human miRNAs printed in the
microarray showed minimal basal expression in wild-type HCT-116 cells and
were upregulated more than 3-fold in DKO cells (FIG. 1A). Of these
significantly upregulated miRNAs, five of them were embedded in a
canonical CpG island (FIG. 1A).

[0224]Bisulfite genomic sequencing analyses of multiple clones of the
original HCT-116 cells demonstrated dense CpG island hypermethylation for
miRNA-124a, miRNA-517c and miR-373 (FIGS. 1B, 3A, 3B, 4A and 4B). Because
we wanted to focus in the cancer-specific DNA methylation changes, we
analyzed by bisulfite genomic sequencing the DNA methylation status of
these miRNA in normal colon tissues (n=10), to exclude tissue-specific
methylation patterns. miRNA-517c and miRNA-373 were found to be densely
methylated in normal colon tissues (FIGS. 3A and 3B), whilst miR-124a
embedded CpG island was always unmethylated (FIGS. 1B, 4A and 4B). In the
case of miR124, this miRNA is represented in three genomic loci
miR-124a-1 (8p23.1), miR-124a-2 (8q12.3) and miR-124a-3 (20q13.33) and in
all cases the corresponding CpG island was methylated in HCT-116 and
unmethylated in normal colon (FIGS. 1B, 1C and FIGS. 4A and 4B). The
presence of miR-124a hypermethylation was not a feature of this
particular cell line, but analyzing a large set of primary human
colorectal tumors was observed in 75% (42 of 56) of patients (FIG. 5D).
The DNA methylation analyses of a comprehensive collection of human
cancer cell lines (n=29) and primary samples (n=115) from breast and lung
carcinomas, leukemias and lymphomas also demonstrated a frequent presence
of miR-124a hypermethylation (FIG. 5D). However, miR-124 methylation was
absent in neuroblastomas (n=22) and sarcomas (n=15) (FIG. 5). Thus,
miR-124a became our prime candidate for a hypermethylation-cancer
specific event.

[0225]To effectively demonstrate the epigenetic silencing of miR-124a in
cancer cells, we first mapped by RACE (Supplementary Methods) the
transcriptional start site of miR-124a and it was found indeed in the
analyzed CpG island (FIG. 1B). Most important, RT-PCR analyses
demonstrated that precursor and mature miR-124a expression was absent in
HCT-116 cells with CpG island hypermethylation and restored in HCT-116
cells treated with the DNA demethylating agent and in DKO cells (FIGS. 1D
and E). The association between miR-124 methylation and loss of
expression was also found in cancer cell lines from other tumor types
(FIGS. 5A and D). Because DNA methylation-silencing is closely linked to
histone modifications to determine active vs inactive gene expression, we
analyzed by chromatin immunoprecipitation the histone modification
pattern and the binding of the transcriptional repressors methyl-CpG
binding domain proteins (MBDs) to the miR-124a CpG island. The presence
of miR-124a CpG island hypermethylation was accompanied by the absence of
histone modification marks associated with gene activation such as
histone H3 and H4 acetylation and trimethylation of lysine 4 of histone
H3, and occupancy by MBDs, such as MeCP2 and MBD2 (FIG. 1F). The
induction of miR-124a CpG island DNA hypomethylation events by the
demethylating agent or in DKO cells was associated with the emergence of
the histone marks of active transcription, and a release of binding by
MBDs (FIG. 1F).

[0226]Finally, to determine whether the epigenetic silencing of miR-124a
had functional cancer relevance to escape the putative tumour suppressor
function of miR-124a, we examined its impact in the regulation of
presumed target genes with oncogenic capacity. Using computational
prediction for miR-124a target genes (Supplementary Methods), we observed
that cyclin D kinase 6 (CDK6) was one of the best potential targets for
miR-124a. CDK6 is involved in cell cycle progression and differentiation
(14) and it constitutes an attractive target for the development of
anti-cancer compounds (15). The first validation that miR-124a can target
CDK6 arise from the expression studies of all the previously described
colon cancer cells. Using CDK6 western-blot analyses we observed that
whilst the original HCT-116 cells with miR-124a methylation-associated
silencing strongly expressed CDK6, the cells treated with the
demethylating agent or the DKO cells showed CDK6 downregulation (FIG.
2A). On the other hand, there was no difference in the CDK6 mRNA
expression levels in any of the described cells (FIG. 2B), suggesting
translational inhibition of CDK6 by miR-124a, rather than mRNA
degradation or transcriptional repression. Most important, a functional
link was established by performing a luciferase reporter assay with a
vector containing the CDK6 wild-type (WT) putative 3' UTR target site and
a mutant form (MUT), in different contexts of miR-124a expression.
Luciferase activity of miR-124a expressing-DKO cells transfected with
CDK6-WT was significantly lower than DKO cells transfected with CDK6-MUT
(p=0.031). In contrast, the luciferase activities of miR-124a-non
expressing-HCT-116 cells transfected with CDK6-WT and CDK6-MUT showed no
measurable differences (FIG. 2D). However, when we co-transfected
miR-124a, luciferase activity of HCT-116 CDK6-WT transfected cells was
significantly lower than CDK6-MUT (p=0.002) (FIG. 2D). To further confirm
the targeting of CDK6 by miR-124a, HCT-116 cells were transfected with
miR-124a precursor molecules, which are designed to mimic endogenous
miR-124a and directly enter the miRNA processing pathway. Overexpression
of miR-124a induced a reduction of CDK6 protein level (FIG. 2E). Most
important, the miR-124a transfection diminished the phosphorylation of Rb
in the residues 807 and 811, the targets of CDK614. The induction of
endogenous miR-124a in HCT-116 cells by the demethylating drug or in DKO
cells also reduced Rb phosphorylation (FIG. 2F). Remarkedly, the
described targeting of CDK6 by miR-124a was also observed in human
primary tumors. An immunostaining analyses of CDK6 expression in lung
cancer patients (n=27), demonstrated that miR-124a hypermethylation was
associated with strong expression of CDK6 and Rb-phosphorylation
(p=0.045) (FIG. 2G). All these data indicate that CDK6 is targeted by
miR-124a and that epigenetic silencing of miR-124a in cancer cells leads
to CDK6 upregulation.

[0227]In summary, we have demonstrated that one mechanism accounting for
the observed downregulation of miRNAs in human cancer is CpG island
hypermethylation, in a similar manner that for classical tumor suppressor
genes. We provide an illustrative example in colon cancer with the
epigenetic silencing of miR-124a and its functional consequences for CDK6
activity. Most important, miRNA function can be restored by erasing DNA
methylation, a finding that may have significant consequences for cancer
patients undergoing treatment with DNA demethylating drugs in the
clinical arena.

[0244]Total RNA was isolated from HCT-116 and DKO cells by Trizol
(Invitrogen) extraction according to the manufacturer's instructions.
miRNA microarray profiling was performed as described by Miska et al (1).
In brief, 5 μg of total RNA was used for each hybridization. miRNA
expression levels were normalized by three different artificial miRNA
spikes. Microarray probes were oligonucleotides with sequences
complementary to microRNAs. Each probe was modified with a free amino
group linked to its 5' terminus through a 6-carbon spacer (IDT) and was
printed onto amine-binding slides (CodeLink, Amersham Biosciences).
Control probes contained two internal mismatches resulting in either
C-to-G or T-to-A changes. Printing and hybridization were done using the
protocols from the slide manufacturer with the following modifications:
the oligonucleotide concentration for printing was 20 μM in 150 mM
sodium phosphate pH 8.5, and hybridization was at 50° C. for 6 h.
Printing was done using a MicroGrid TAS II arrayer (BioRobotics) at 50%
humidity. DNA methylation analyses. The CpG Island Searcher Program (2)
was used to determine which miRNAs were embedded in a CpG island. The DNA
methylation status was established by PCR analysis of bisulfite-modified
genomic DNA, which induces chemical conversion of unmethylated, but not
methylated, cytosine to uracil, using two procedures. First, methylation
status was analyzed by bisulfite genomic sequencing of both strands of
the corresponding CpG islands. The second analysis used methylation
specific PCR using primers specific for either the methylated or modified
unmethylated DNA. DNA from normal lymphocytes treated in vitro with Sssl
methyltransferase was used as a positive control for methylated alleles.
DNA from normal lymphocytes was used as a positive control for
unmethylated alleles.

[0245]5' Rapid Amplification of cDNA Ends (RACE) system was performed
using Invitrogen kit and according to the manufacturer's instruction.
Briefly, 5 μg of brain total RNA was reverse transcribed into cDNA
using SuperScript® II RT reverse transcriptase and specific reverse
primers (GSP1):

[0246]After that, cDNAs were amplified by PCR using Elongase®
Amplification System (Invitrogen) and others specific primers (GSP2 or
GSP3 for nested amplification in the cases of miR-124a1 and miR-124a2).
The PCR primers are as follows:

[0248]GAPDH was used as internal control. PCR products were analyzed in a
3% agarose gel. Realtime PCR analysis of miRNAs was performed as
previously described (Biotechniques 39:519-525). In brief, total RNA was
isolated from cell lines or tissues using Trizol reagent and the small
RNA fraction was further isolated using the miRVANA kit (Ambion). 500 ng
of the small RNA fraction was polyadenylated by poly(A) polymerase (PAP,
Ambion) at 37° C. for 1 hr in a 6 uL reaction. After
phenol-chloroform extraction and ethanol precipitation the polyadenylated
small RNAs were reversed transcribed according to the manufacturer's
protocols using 200U SuperScript-II Reverse Transcriptase (RT,
Invitrogen) and 0.5 ug poly(T) adapter (GCG AGC ACA GM TTA ATA CGA CTC
ACT ATA GGT TTT TTT TTT TTV N) (SEQ ID NO:36). The small cDNAs were then
treated with RNAse H at 37° C. for 1 hr and finally diluted 240
fold. Negative controls were included in both PAP and RT reactions.
Moreover, two additional RT reactions were performed using half and
double the input of polyadenylated small RNAs to ensure the linearity of
the reverse transcription step. Finally, the melting temperature for each
miRNA was experimentally determined from the thermal dissociation curves
using ABI Prism 7900HT (Applied Biosystems, Foster City, Calif., USA).
The real-time PCR reactions were then carried out at the optimal
temperature and typically contained 5 pmol of the gene-specific primer
(identical to the complete miRNA sequence listed in the miRBASE
database), 5 pmol of the common antisense oligonucleotide
(GCGAGCACAGAATTAATACGACTC) (SEQ ID NO: 37), 10 μL SYBR Green PCR
Master Mix (Applied Biosystems) and 6 uL of the diluted cDNAs in a total
volume of 20 μL. The specific sense primers used were

[0249]Each set of PCR reactions included a dilution series of a reference
cDNA to be used for quantification. The following quality control was
applied before data sets were accepted: (1) both PAP and RT negative
controls gave negligible values or displayed dissociation curves that
were significantly different from those in experimental samples, (2) the
realtime PCR values of samples containing RT reactions programmed with
different RNA inputs showed a dose response, (3) the linear fit of the
real-time PCR values of the dilution series was >90%.

Chromatin Immunoprecipitation (ChIP) Assay.

[0250]Standard ChIP assays were performed as previously described (3). In
brief, cells were treated with 1% formaldehyde for 15 min. Then,
chromatin was sheared with a Bioruptor® (Diagenode) to an average
length of 0.4-0.8 kb for this analysis. The following antibodies were
used: anti-MeCP2 (ab3752), anti-MBD2 (ab3754), and anti-trimethyl-K4
histone H3 (ab8580) (ab1220) (Abcam) and anti-acetyl H3 (06-599) and
anti-acetyl H4 (06-598) (Upstate biotechnologies). PCR amplification was
performed in 20 μl with specific primers for each of the analyzed
promoters.

[0252]The miRNA sequences were analyzed using miRBase
(http://microrna.sanger.ac.uk/), University of California at Santa Cruz
Human Genome Browser (http://genome.cse.ucsc.edu/) and Methyl Primer
Express (Applied Biosystems). Detailed information of base pairing
comparison between miR-124a and its target site in the 3'UTR of CDK6 mRNA
is available at Human microRNA Targets (http://www.microrna.org/) and
miRBase Targets (http://microrna.sanger.ac.uk/). The GenBank accession
number of CDK6 mRNA is NM--001259.

[0253]Analyses of CDK6 and phosphorylated Rb expression by Western blot,
immunohistochemistry and RT-PCR. We collected cells by centrifugation and
washed cell pellets twice with phosphate-buffered saline buffer. Total
extracts were fractionated on a SDS-PAGE gel and transferred to a
polyvinylidene difluoride membrane with 45-μm pore size (Immobilon
PSQ, Millipore). The membranes were blocked in 5% milk PBS-T
(phosphate-buffered saline with 0.1% Tween-20) and immunoprobed with
antibodies against CDK6 (1:1000; Cell Signaling) and P-Rb-S807/811
(1:1000; Cell Signaling). An antibody to β-actin (1:5000; Sigma) was
used as a loading control. Immunoreactive bands were visualized using a
chemiluminescent substrate (Amersham) after incubation of the membrane
with secondary antibodies conjugated to horseradish peroxidase.

[0254]Immunohistochemical staining of CDK6 and PRb-S807/811 was performed
using the described above antibodies at a 1:1500 dilution. After antigen
retrieval with citrate buffer, immunodetection was performed by the DAKO
EnVision Visualization Method (DAKO, Glostrup, Denmark), with
diaminobenzidine chromogen as the substrate. For the RT-PCR of CDK6,
total RNA (5 μg) was used for reverse transcription. After incubation
with DNase I (Invitrogen) to eliminate DNA contamination, Superscript III
(Invitrogen) and random hexamers (Promega, Madison, Wis.) were added for
first strand cDNA synthesis. Then PCR was performed with primers specific
for CDK6 mRNA (forward, 5'-GCC TAT GGG MG GTG TTC M-3' (SEQ ID NO: 46);
reverse, 5'-CAC TCC AGG CTC TGG MC TT-3') (SEQ ID NO: 47).

[0255]Transfection with miR-124a precursor molecules and luciferase
assays. miR-124a precursor molecules and negative control miRNA were
purchased from Ambion. Experiments involving transient transfections of
miRNAs were carried out with oligofectamine (Invitrogen) using 100 nM RNA
duplexes. The cells were collected 48 h after transfection and the
expression of CDK6 and P-Rb-S807/811 were analyzed by Western Blot and
RT-PCR, as described above. For Luciferase experiments the luciferase
reporter assay system (Ambion) was used. Luciferase constructs were made
by ligating oligonucleotides containing the wild-type or mutant putative
target site of the CDK6 3'UTR into the multicloning site of the p-MIR
Reporter Luciferase vector (Ambion). Cells were cotransfected using
Lipofectamine 2000 (Invitrogen) with 0.4 μg of firefly luciferase
reporter vector containing the wild type or mutant oligonucleotides, 0.02
μg pGal control vector, and 100 ng of miR-124a precursor. Luciferase
activity was measured 48 hours after transfection using β-gal for
normalization.

[0259]The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described herein will become apparent to
those skilled in the art from the foregoing description and accompanying
figures. Such modifications are intended to fall within the scope of the
appended claims. Moreover, all embodiments described herein are
considered to be broadly applicable and combinable with any and all other
consistent embodiments, as appropriate.

[0260]Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.